WO2023034904A1 - Cellules recombinantes pour le traitement de maladies associées à l'acide urique et leurs procédés d'utilisation - Google Patents

Cellules recombinantes pour le traitement de maladies associées à l'acide urique et leurs procédés d'utilisation Download PDF

Info

Publication number
WO2023034904A1
WO2023034904A1 PCT/US2022/075820 US2022075820W WO2023034904A1 WO 2023034904 A1 WO2023034904 A1 WO 2023034904A1 US 2022075820 W US2022075820 W US 2022075820W WO 2023034904 A1 WO2023034904 A1 WO 2023034904A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
gene
sequence
host cell
cell
Prior art date
Application number
PCT/US2022/075820
Other languages
English (en)
Inventor
Dylan Alexander CARLIN
Sean COTTON
Lucas HARTSOUGH
Vincent M. Isabella
Rishi Jain
Theodore Carlton MOORE, III
Mylene PERREAULT
Pichet PRAVESCHOTINUNT
Seth RITTER
Mehmet TATLI
Original Assignee
Synlogic Operating Company, Inc.
Ginkgo Bioworks, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Synlogic Operating Company, Inc., Ginkgo Bioworks, Inc. filed Critical Synlogic Operating Company, Inc.
Priority to AU2022340814A priority Critical patent/AU2022340814A1/en
Publication of WO2023034904A1 publication Critical patent/WO2023034904A1/fr

Links

Classifications

    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
    • C12N9/0046—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7) with oxygen as acceptor (1.7.3)
    • C12N9/0048—Uricase (1.7.3.3)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y107/00—Oxidoreductases acting on other nitrogenous compounds as donors (1.7)
    • C12Y107/03—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with oxygen as acceptor (1.7.3)
    • C12Y107/03003—Factor-independent urate hydroxylase (1.7.3.3), i.e. uricase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Said XML copy, created on September 1, 2022, is named 126046-07120_SL.XML and is 751,987 bytes in size.
  • Increased levels of uric acid from excess purines may accumulate in the tissues, forming crystals, and may occur when blood uric acid levels rise to above about 7 mg/dL.
  • Hyperuricemia is a risk factor for the development of chronic conditions such as gout, renal dysfunction, hypertension, diabetes and obesity (Maiuolo et al. Int J of Card (2016) 213:15, Kushiyama et al. Mediators Inflamm. (2016) 2016:86031).
  • Gout is a form of inflammatory arthritis characterized by sudden, severe attacks of pain and swelling in the joints. The symptoms of gout are due to high serum levels of uric acid, commonly related to a high purine-rich diet involving higher consumption of meat and fish.
  • uricolytic pegloticase a mammalian recombinant uricase conjugated to monomethoxypoly (ethylene glycol).
  • pegloticase treatment demonstrates moderate clinical outcomes, it requires recurrent patient intervention and has a high risk for developing infusion reactions with severe consequences (Calabrese et al.
  • the present disclosure provides host cells (e.g., a bacterial cell, a plant cell, an algal cell, a fungal cell, a yeast cell, or an animal cell), pharmaceutical compositions thereof, and methods of modulating and treating diseases associated with uric acid, such as hyperuricemia and gout.
  • the host cells disclosed herein have been constructed to express one or more uric acid catabolism enzyme(s), e.g., uric acid degrading enzymes, such as a uricase.
  • the host cell further comprises an importer, e.g., uric acid or urate importer.
  • the host cells e.g., bacterial cells
  • the host cells further comprise other genetic circuitry in order to guarantee the safety and non-colonization of a subject that is administered the recombinant bacteria, such as auxotrophies, kill switches, etc. These recombinant bacteria are safe and well tolerated and augment the innate activities of the subject’s microbiome to achieve a therapeutic effect.
  • a host cell comprising a heterologous uricase gene operably linked to a first promoter, wherein the heterologous uricase gene encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NOs: 219, 218, 219, 229, 230, 261, 263, 265, 267, 269, 271, or 273.
  • a host cell comprising a heterologous uricase gene operably linked to a first promoter, wherein the heterologous uricase gene comprises a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NOs: 209, 210, 226, 227, 260, 262, 264, 266, 268, 270, or 272.
  • the host cell further comprises a heterologous gene encoding a urate importer.
  • the heterologous gene encoding the urate importer encodes a polypeptide comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of any one of SEQ ID NOs: 220, 10, 221, 222, 246- 257, or 259.
  • the heterologous gene encoding the urate importer comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of any one of SEQ ID NOs: 5, 211-213, 234-245, or 258.
  • the heterologous gene encoding the urate importer is operably linked to the first promoter or a second promoter.
  • the host cell does not comprise a heterologous gene encoding a urate importer.
  • a host cell comprising a heterologous urate importer gene operably linked to a first promoter, wherein the heterologous urate importer gene encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of any one of SEQ ID NOs: 220-222, 246-257, or 259.
  • the sequence of the urate importer comprises one or more amino acid substitutions relative to the sequence of SEQ ID NO: 10. In one embodiment, the one or more substitutions are at positions corresponding to position 275 and/or 346 in SEQ ID NO: 10. In one embodiment, the urate importer comprises: an alanine (A) at a position corresponding to position 346 in SEQ ID NO: 10; and/or a methionine (M) at a position corresponding to position 275 in SEQ ID NO: 10.
  • A alanine
  • M methionine
  • a host cell that comprises a heterologous polynucleotide encoding a urate importer, wherein the sequence of the urate importer comprises an amino acid substitution relative to the sequence of SEQ ID NO: 10, optionally wherein the substation is at the position corresponding to position 275 in SEQ ID NO: 10.
  • the urate importer comprises a methionine (M) at the position corresponding to position 275 in SEQ ID NO: 10.
  • a host cell comprising a heterologous gene encoding a urate importer operably linked to a first promoter, wherein the gene comprises a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NOs: 211-213, 234-245, or 258.
  • the host cell further comprises a heterologous uricase gene.
  • the heterologous uricase gene is operably linked to the first promoter or a second promoter.
  • the heterologous uricase gene encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NOs: 8, 218, 219, 228-230, 261, 263, 265, 267, 269, 271, or 273.
  • the heterologous uricase gene comprises a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NOs: 3, 209, 210, 225-227, 260, 262, 264, 266, 268, 270, or 272.
  • the host cell comprises three copies of the heterologous uricase gene. [019] In one embodiment, the host cell comprises three copies of the heterologous gene encoding the urate importer. [020] In one embodiment, the first promoter, the second promoter, or the first promoter and the second promoter are inducible promoters, constitutive promoters, or an inducible promoter and a constitutive promoter. In one embodiment, the inducible promoter is an IPTG-inducible promoter. In one embodiment, the inducible promoter is a Ptac promoter. In one embodiment, the Ptac promoter comprises a sequence of SEQ ID NO: 1115.
  • the inducible promoter is directly or indirectly induced by temperature.
  • the promoter directly or indirectly induced by temperature comprises a sequence of any one of SEQ ID NOs: 233, 309, 313, and 316.
  • the host cell further comprises a gene sequence encoding a mutant repressor of the Pr/Pl promoter.
  • the gene sequence encoding a mutant repressor comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of any one of SEQ ID NOs: 233 and 314.
  • the heterologous uricase gene, the heterologous gene encoding the urate importer, or both the heterologous uricase gene and the heterologous gene encoding the urate importer are located on a plasmid or a chromosome in the cell.
  • the host cell is a bacterial cell, a plant cell, an algal cell, a fungal cell, a yeast cell, or an animal cell. In one embodiment, the host cell is a bacterial cell.
  • the bacterial cell is a probiotic bacterial cell. In one embodiment, the bacterial cell is of the species Escherichia coli strain Nissle.
  • the host cell is a yeast cell.
  • the yeast cell is a Saccharomyces cell, a Yarrowia cell, a Komagataella cell, or a Pichia cell.
  • the Saccharomyces cell is a Saccharomyces cerevisiae cell.
  • the yeast cell is a Yarrowia cell.
  • the host cell further comprises an insertion, deletion or mutation of an endogenous phage gene.
  • the insertion, deletion or mutation is a deletion of the endogenous phage gene comprising a sequence of SEQ ID NO: 1064.
  • the host cell further comprises a modified endogenous colibactin island.
  • the modified endogenous colibactin island comprises one or more modified clb sequences selected from the group consisting of clbA (SEQ ID NO: 1065), clbB (SEQ ID NO: 1066), clbC (SEQ ID NO: 1067), clbD (SEQ ID NO: 1068), clbE (SEQ ID NO: 1069), clbF (SEQ ID NO: 1070), clbG (SEQ ID NO: 1071), clbH (SEQ ID NO: 1072), clbI (SEQ ID NO: 1073), clbJ (SEQ ID NO: 1074), clbK (SEQ ID NO: 1075), clbL (SEQ ID NO: 1076), clbM (SEQ ID NO: 1077), clbN (SEQ ID NO: 1078),
  • the modified endogenous colibactin island comprises a deletion of clbA (SEQ ID NO: 1065), clbB (SEQ ID NO: 1066), clbC (SEQ ID NO: 1067), clbD (SEQ ID NO: 1068), clbE (SEQ ID NO: 1069), clbF (SEQ ID NO: 1070), clbG (SEQ ID NO: 1071), clbH (SEQ ID NO: 1072), clbI (SEQ ID NO: 1073), clbJ (SEQ ID NO: 1074), clbK (SEQ ID NO: 1075), clbL (SEQ ID NO: 1076), clbM (SEQ ID NO: 1077), clbN (SEQ ID NO: 1078), clbO (SEQ ID NO: 1079), clbP (SEQ ID NO: 1080), clbQ (SEQ ID NO:
  • the host cell further comprises an auxotrophy.
  • the auxotrophy is a ⁇ dapA auxotrophy.
  • the bacterial cell does not comprise a gene encoding for antibiotic resistance.
  • the host cell comprises a further modification or deletion of a cytochrome oxidase, e.g., a modification or deletion of appB (SEQ ID NO: 214), a modification or deletion of appC (SEQ ID NO: 215), and/or a modification or deletion of both appB and appC (SEQ ID NOs: 214 and 215, respectively).
  • a host cell wherein the host cell is a recombinant bacterial cell, comprising: three copies of a heterologous uricase gene operably linked to a first promoter, wherein the heterologous uricase gene encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 219, three copies of a heterologous urate importer gene operably linked to a second promoter, wherein the heterologous urate importer gene encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 222, a phage deletion, a deletion of an endogenous colibactin island, and a ⁇ dapA auxotrophy.
  • the cell is capable of reducing levels of uric acid in vitro cell culture by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, or at least about 85% in about 30 minutes. In one embodiment, the cell is capable of reducing levels of uric acid in vitro cell culture by at least about 90%, at least about 95%, or at least about 100% in about 90 minutes.
  • the cell is capable of degrading uric acid at a rate of at least about 0.1 ⁇ mol/1x10 9 cells/hr, at least about 0.2 ⁇ mol/1x10 9 cells/hr, at least about 0.3 ⁇ mol/1x10 9 cells/hr, at least about 0.4 ⁇ mol/1x10 9 cells/hr, at least about 0.5 ⁇ mol/1x10 9 cells/hr, at least about 0.6 ⁇ mol/1x10 9 cells/hr, at least about 0.7 ⁇ mol/1x10 9 cells/hr, at least about 0.8 ⁇ mol/1x10 9 cells/hr, at least about 0.9 ⁇ mol/1x10 9 cells/hr, or at least about 1.0 ⁇ mol/1x10 9 cells/hr in in vitro cell culture.
  • the cell is SYN7960, SYN7229, SYN8581, SYN8592, SYN8634, or SYN8669.
  • a pharmaceutical composition comprising any one of the host cell disclosed herein and a pharmaceutically acceptable carrier.
  • a method for treating a disease associated with uric acid in a subject comprising administering the pharmaceutical composition of claim 50 to the subject, thereby treating the disease in the subject.
  • a method for reducing a level of uric acid in a subject comprising administering to the subject the pharmaceutical composition of claim 50, thereby reducing the level of uric acid in the subject.
  • the subject has hyperuricemia or gout.
  • the host cell is a bacterial cell, and wherein the pharmaceutical composition comprises about 5x10 10 , about 1x10 11 , about 5x10 11 , or about 1x10 12 live recombinant bacterial cells/mL.
  • the pharmaceutical composition comprises about 1x10 11 live recombinant bacterial cells/mL.
  • the pharmaceutical composition comprises about 5x10 11 live recombinant bacterial cells/mL.
  • the levels of uric acid in the subject are reduced by at least about 1-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5- fold, at least about 6-fold, at least about 7-fold, or at least about 8-fold.
  • the levels of uric acid in a subject are reduced by at least 1-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, or at least about 8-fold 6 hours after administration of the pharmaceutical composition when compared to prior to administration.
  • the pharmaceutical composition is administered orally.
  • the subject is a human subject.
  • a level of allantoin is measured in the subject prior to administration and after administration, and an increased level allantoin in the subject after administration is an indication that the treatment is effective.
  • the level of allantoin after administration is increased by at least about 10%, 20%, 25%, 50%, 75%, or 100% as compared to the level of allantoin prior to administration. In one embodiment, the level of allantoin 6 hours after administration is increased by at least about 10%, 20%, 25%, 50%, 75%, or 100% as compared to the level of allantoin prior to administration. [047] In another aspect, disclosed herein is a method comprising culturing any one of the host cell disclosed herein. In another aspect, disclosed herein is a method of metabolizing uric acid comprising culturing any one of the host cell disclosed herein.
  • a non-naturally occurring urate importer comprising a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 221.
  • a non-naturally occurring nucleic acid encoding a urate importer wherein the nucleic acid comprises a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 212.
  • a vector comprising any one of the non-naturally occurring nucleic acid disclosed herein.
  • an expression cassette comprising the non-naturally occurring nucleic acid provided herein.
  • the heterologous gene encoding the uric acid catabolism enzyme is located on a plasmid or a chromosome in the cell, e.g., bacterial cell, e.g., recombinant bacterial cell.
  • the plasmid is a low-copy number plasmid (e.g., 3-5 copies/cell), a medium-copy number plasmid (e.g., 10-15 copies/cell), or a high copy-number plasmid (e.g., 50 or more copies/cell).
  • the heterologous gene encoding the urate importer is located on a plasmid or a chromosome in the cell.
  • the plasmid is a low-copy number plasmid (e.g., 3-5 copies/cell), a medium-copy number plasmid (e.g., 10-12 copies/cell), or a high copy- number plasmid (e.g., 50 or more copies/cell).
  • a composition comprising the cell, e.g., bacterial cell, e.g., recombinant bacterial cell.
  • the recombinant bacterial cell has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% viability. In one embodiment, the recombinant bacterial cell has at least about 90% viability.
  • viability is measured using a cell dye penetration/extrusion assay. Methods for measuring cell viability are described, for example, in PCT/US2020/030468 (filed on April 29, 2020 and published as WO2020/223345), the entire contents of which are expressly incorporated herein by reference in their entirety.
  • the administration prevents formation of kidney stones in the subject.
  • the subject is fed a meal within one hour of administering the pharmaceutical composition. In another embodiment, the subject is fed a meal concurrently with administering the pharmaceutical composition. In one embodiment, the pharmaceutical composition is administered orally. In one embodiment, the subject is a human subject.
  • a method of manufacturing the recombinant bacterial cell comprising growing the recombinant bacterial cell in a fermenter vessel in the presence of glucose or glycerol to produce a population of recombinant bacterial cells, adding an inducer to the fermenter vessel to induce expression of the first promoter and/or the second promoter, harvesting the population of recombinant bacterial cells by centrifugation, and resuspending the population of recombinant bacterial cells in a buffer, wherein population of recombinant bacterial cells has a viability of at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.
  • the fermenter vessel is an AMBR fermenter or a 3L fermenter.
  • the buffer is water or a PKU buffer.
  • the method further comprises measuring viability of the population of recombinant bacterial cells. In one embodiment, viability of the population of recombinant bacterial cells is measured using a cell dye penetration/extrusion assay. Brief Description of the Drawings [056] FIG.1 depicts that purine degradation leads to production of uric acid. Guanine monophosphate (GMP) is converted to guanosine by a nucleotidase.
  • GMP Guanine monophosphate
  • nucleosides inosine and guanosine are further converted to purine base hypoxanthine and guanine, respectively, by purine nucleoside phosphorylase (PNP).
  • Hypoxanthine is then oxidized to form xanthine by xanthine- oxidase (XO), and guanine is deaminated to form xanthine by guanine deaminase.
  • Xanthine is again oxidized by xanthine oxidase to form the final product, uric acid.
  • adenosine is likely important.
  • FIG.2 depicts degradation of uric acid under anaerobic and microaerobic conditions in E. coli by two putative oxidoreductases (aegA and ygfT) (K Iwadate and J Kato, J Bacteriol, 201:11 (2019)).
  • FIGs.3A and 3B depict transportation of uric acid into E. coli by the proton symporter YgfU.
  • FIG.3A shows the schematic of transporter.
  • FIG.3B demonstrates both active and selective transport of uric acid (K Papakostas and S Frillingos, J Biol Chem, 287(19): 15684-95, (2012)).
  • FIG.4 depicts a schematic of an engineered bacterial cell with 1) a construct expressing UacT, and 2) a construct expressing uricase.
  • FIGs.5A and 5B are graphs showing uric acid consumption.
  • FIG.5A depicts a line graph comparing uric acid consumption over time by uricase, SYN094 (E. coli Nissle control) lysate, SYN094 (E. coli Nissle control) whole cell, SYN7229 (C. utilis uricase (SEQ ID NO: 228), E. coli MG1655 transporter (SEQ ID NO: 10)) lysate, and SYN7229 (C. utilis uricase (SEQ ID NO: 228), E. coli MG1655 transporter (SEQ ID NO: 10))whole cell.
  • FIG.5B depicts a bar graph of total uric acid consumption over four hours by uricase, SYN094 (E.
  • FIGs.6A and 6B depict levels of uric acid (UA) in urine after administration of SYN7229 (C. utilis uricase (SEQ ID NO: 228), E.
  • FIGs.6C and 6D depict levels of uric acid in urine after administration of SYN7229 (C. utilis uricase (SEQ ID NO: 228), E. coli MG1655 transporter (SEQ ID NO: 10)) (glycerol-process) and SYN094 (E. coli Nissle control) (glucose-process) in an acute mouse model of hyperuricosuria.
  • FIG.7 depicts results from a metagenomics library screen identifying C. utilis uricase and other uricase candidates.
  • Uricase activity was measured by measuring the disappearance of uric acid at 290 nm.
  • induced EcN cells were brought to OD600 of 1 using M9 media containing 2.5 mM uric acid in a 96 deep-well plate. The plate was incubated at 37 o C at 1000 rpm for 4 hours. After 4 hours, the uric acid level in the supernatant was measured at 290 nm on the plate reader. The UA level goes to below zero as it was OD normalized and calculated by this formula: 2.5 mM - ([UA] consumed/O.D.600).
  • FIG.8 depicts uric acid consumption by E. coli strains in media.
  • Strains 851714 (C. utilis v1 uricase-Ptet (SEQ ID NO: 228), E. ictaluri transporter-PlacIO (SEQ ID NO: 220)); 851573 (A. globiformis uricase (SEQ ID NO: 218)); 851774 (Mus musculus uricase (SEQ ID NO: 230), E. ictaluri transporter (SEQ ID NO: 220)); 851801 (C. utilis v2 uricase (SEQ ID NO: 229), E. ictaluri transporter (SEQ ID NO: 220)); and 851796 (C.
  • FIG.9A depicts uric acid consumption by E. coli strains with uricase in media. Strains: 870791 (C. californicus uricase (SEQ ID NO: 219)); 776000 (C. utilis uricase-Ptet (SEQ ID NO: 228)); and 890735 (C. utilis uricase-PlacIO (SEQ ID NO: 228)).
  • FIG.9B depicts uric acid consumption by E. coli strains with uricase and uric acid transporter in media. Strains: 851796 (SYN7957) (C. californicus uricase (SEQ ID NO: 219), E.
  • FIG.10 depicts top metagenomics hits derived from human gut bacteria, N-fixing soil bacteria, and fish pathogen. Data indicating activity of recombinant urate transporters under different conditions. Briefly, an E.
  • FIG.11 depicts uric acid consumption by E. coli at 37 °C with shaking or without shaking (microaer).
  • FIG.12 depicts uric acid consumption by E. coli at 37°C with and without shaking (microaer).
  • SYN7229 C. utilis uricase (SEQ ID NO: 228), E. coli MG1655 transporter (SEQ ID NO: 10)
  • SYN7896 C. utilis uricase-Ptet (SEQ ID NO: 228), E. ictaluri transporter-PlacIO (SEQ ID NO: 220)
  • SYN7897 C. utilis uricase-Ptet (SEQ ID NO: 228), E. tarda transporter-PlacIO (SEQ ID NO: 222)
  • SYN7898 C. utilis uricase (SEQ ID NO: 228), E. coli F275M transporter (engineered; SEQ ID NO: 221)).
  • FIG.13 depicts uric acid consumption by E. coli at 37°C with and without shaking (microaer).
  • Strains SYN7957 (C. californicus uricase (SEQ ID NO: 219), E. ictaluri transporter (SEQ ID NO: 220)); SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)); and SYN7997 (C. utilis uricase-PlacIO (SEQ ID NO: 228), E. tarda transporter-PlacIO (SEQ ID NO: 222)).
  • FIG.14 depicts uric acid consumption by E.
  • FIG.15A depicts uric acid consumption by E.
  • SYN094 E. coli Nissle control
  • SYN7957 C. californicus uricase (SEQ ID NO: 219), E. ictaluri transporter (SEQ ID NO: 220)
  • SYN7959 890902
  • SYN7960 C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)
  • SYN7997 C.
  • FIG.15B depicts allantoin production by E. coli in SIF. Strains: SYN094 (E. coli Nissle control); SYN7957 (C.
  • californicus uricase SEQ ID NO: 219), E. ictaluri transporter (SEQ ID NO: 220)); SYN7959 (890902) (C. utilis uricase-PlacIO (SEQ ID NO: 228), E. ictaluri transporter-PlacIO (SEQ ID NO: 220)); SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)); SYN7997 (C. utilis uricase-PlacIO (SEQ ID NO: 228), E. tarda transporter-PlacIO (SEQ ID NO: 222)); SYN8012 (C.
  • FIG.16 depicts uric acid consumption by E. coli in M9 media.
  • Strains SYN094 (E. coli Nissle control); SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)); SYN8196 (C.
  • FIG.17A depicts decreased urinary 15N-uric acid recovered from non-human primates (NHP) after treatment with SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)) compared to vehicle.
  • FIG.17B depicts decreased urinary endogenous uric acid recovered from non-human primates (NHP) after treatment with SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)) compared to vehicle.
  • FIG.18A depicts decreased 15N-uric acid in feces recovered from non-human primates (NHP) after treatment with SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)) compared to vehicle.
  • FIG.18B depicts decreased endogenous uric acid in feces recovered from non-human primates (NHP) after treatment with SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)) compared to vehicle.
  • FIG.19A depicts decreased urinary 15N-uric acid recovered from non-human primates (NHP) after treatment with SYN7229 (C. utilis uricase (SEQ ID NO: 228), E. coli MG1655 transporter (SEQ ID NO: 10)) compared to vehicle and SYN094 (E. coli Nissle control).
  • FIG.19B depicts decreased urinary endogenous uric acid recovered from non-human primates (NHP) after treatment with SYN7229 (C. utilis uricase (SEQ ID NO: 228), E. coli MG1655 transporter (SEQ ID NO: 10)) compared to vehicle and SYN094 (E. coli Nissle control).
  • FIG.20 depicts uric acid consumption and allantoin production by E. coli. Consumption of uric acid and production of allantoin in vitro.1E9 cells were added to minimal media containing 1mM UA and 0.5% glucose and incubated at 37 ⁇ C for 2hrs. Strains: SYN-GOUT (SYN7960 (C.
  • FIG.21 depicts uric acid consumption by E. coli at 37 °C without shaking.
  • FIG.22 depicts allantoin production by E. coli in M9 media without shaking.
  • Strains SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)); SYN8197 (C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222)); SYN8278 (C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222), C.
  • FIG.23 depicts integration of uricase and transporter genes with attP2, attP5, and/or attP7 integration vectors. Promoter: temperature-sensitive system.
  • FIG.24 is a schematic of integration of multiple uricase and transporter genes in the E. coli chromosome. Chassis with auxotrophy, phage and colibactin removed. Enzymes all 37C-inducible. Three copies of each enzyme + transporter tandem at each LP site.
  • FIG.25A depicts uric acid consumption by E. coli at 37 °C without shaking. Strains: SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)); SYN8581 (C.
  • FIG.25B depicts uric acid consumption by E.
  • Plasmid SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222))
  • 1 copy integrant SYN8197 (C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222))
  • SYN094 E. coli Nissle control
  • FIG.26A depicts in vitro uric acid consumption by E. coli at 37 °C without shaking.
  • Strains SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)); SYN8581 (C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222); 1 copy each, Pr/L promoter and cI38 repressor; ⁇ clb ⁇ dapA ⁇ ); SYN8592 (C.
  • californicus uricase integrated (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222); 2 copies each; ⁇ clb ⁇ dapA ⁇ ); SYN8634 (C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222); 3 copies each; ⁇ clb ⁇ dapA ⁇ ); and SYN8669 (C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222); 3 copies each; cured of antibiotic resistance; ⁇ clb ⁇ dapA ⁇ ).
  • FIG.26B depicts consumption of uric acid in vitro.1E9 cells were added to minimal media containing 1mM UA (no carbon source) and incubated at 37 ⁇ C for 2hrs. Optimization involved switching from a chemically-induced to an environmentally-sensitive promoter and altering the ribosomal binding site (RBS) of the uricase gene by increasing the predicted translation initiation rate.
  • californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222); 1 copy each, Pr/L promoter and cI38 repressor; ⁇ clb ⁇ dapA ⁇ ) ; “1 st Opt.”); SYN8592 (C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222); 2 copies each; ⁇ clb ⁇ dapA ⁇ ); “2 nd Opt.”); SYN8634 (C. californicus uricase (integrated) (SEQ ID NO: 219), E.
  • FIG.27 depicts SYN-GOUT (SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222))) allantoin production in low oxygen conditions. Strain activity as measured by production of allantoin under various dissolved oxygen (DO) conditions.1E9 cells were added to minimal media containing 5mM UA and 0.5% glucose and incubated at 37 ⁇ C over 3hrs.
  • DO dissolved oxygen
  • FIG.28 depicts SYN-GOUT (SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222))) reducing levels of endogenous uric acid in urinary output in non-human primates. Demonstration of urinary uric acid lowering in nonhuman primates dosed with 15N-labeled uric acid. Endogenous (and labeled, data not shown) urinary uric acid output is significantly reduced when NHPs were given an oral gavage of 1E11 cells of SYN- GOUT (SYN7960) compared to both vehicle treated and EcN treated NHP.
  • SYN7960 C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)
  • FIG.29 depicts schematic of SYN8669 (C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222); 3 copies each; cured of antibiotic resistance; ⁇ clb ⁇ dapA ⁇ ).
  • FIG.30 depicts a graph showing uric acid consumption by E. coli at 37 °C without shaking. Strains: SYN7229 (C. utilis uricase (SEQ ID NO: 228), E. coli MG1655 transporter (SEQ ID NO: 10)); SYN7960 (C.
  • FIG.31A depicts a schematic showing the study design of a mouse uric acid enterocirculation study.
  • FIG.31B depicts a graph showing plasma levels of 15N-uric acid (15N-UA) at the indicated times post oral administration.
  • FIG.31C and FIG.31D are graphs showing plasma (FIG.31C) and small intestinal (FIG.
  • FIG.32A and FIG.32B are graphs showing the amounts of uric acid (FIG.32A) and allantoin (FIG.32B) in stomach, duodenum, jejunum, ileum, and colon of NHPs. Monkeys were dosed a bolus including peptone, sodium bicarbonate, D5-Phe, and formulation buffer and were sacrificed at 30min post-dose.
  • FIG.33A depicts a schematic showing the design of a study in which non-human primates (NHPs) were administered 15N-UA to transiently elevate uricemia.
  • NHS non-human primates
  • FIG.33B and FIG.33C are graphs showing plasma levels of 15N-uric acid (FIG.33B) and 15N-Allantoin (FIG.33C).
  • FIG.33D and FIG.33E are graphs showing urinary levels of 15N-uric acid (FIG.33D) and 15N-Allantoin (FIG.33E).
  • FIGs.34A-34D are graphs comparing the effect of oral gavage of SYN8669 (C. californicus uricase (integrated) (SEQ ID NO: 219), E.
  • tarda transporter (integrated) SEQ ID NO: 222
  • the present disclosure provides host cells, e.g., recombinant cells (e.g., a bacterial cell, a plant cell, an algal cell, a fungal cell, a yeast cell, or an animal cell), pharmaceutical compositions thereof, and methods of modulating and treating diseases associated with uric acid, such as hyperuricemia and/or gout.
  • host cells e.g., recombinant cells (e.g., a bacterial cell, a plant cell, an algal cell, a fungal cell, a yeast cell, or an animal cell), pharmaceutical compositions thereof, and methods of modulating and treating diseases associated with uric acid, such as hyperuricemia and/or gout.
  • the recombinant cells e.g., bacterial, plant, fungal, or animal cells
  • the recombinant cells have been constructed to comprise genetic circuits composed of, for example, a uricase enzyme and/or a urate transporter to treat disease, as well as (optionally) other circuitry in order to guarantee the safety and non-colonization of the subject that is administered the recombinant cells (e.g., bacterial, plant, fungal or animal cells), such as auxotrophies, kill switches, etc.
  • recombinant cells e.g., bacterial cells
  • auxotrophies e.g., kill switches, etc.
  • the disclosure pertains to a polynucleotide having a sequence which comprises, is or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to any sequence disclosed herein.
  • the disclosure pertains to a cell, e.g., a host cell or a recombinant cell, comprising such a polynucleotide.
  • a recombinant cell e.g., bacterial, plant, fungal or animal cell
  • a recombinant cell has been genetically engineered to comprise a heterologous gene sequence encoding one or more uric acid catabolism enzymes and is capable of processing (e.g., metabolizing) and reducing levels of uric acid.
  • a recombinant cell e.g., bacterial, plant, fungal or animal cell
  • a recombinant cell e.g., bacterial, plant, fungal or animal cell
  • the genetically engineered cells e.g., bacterial, plant, fungal or animal cells
  • pharmaceutical compositions comprising the bacterial cells disclosed herein may be used to convert excess uric acid into non-toxic molecules in order to treat and/or prevent diseases associated with uric acid, such as hyperuricemia and/or gout.
  • the term “cell” refers to any cell, e.g., bacterial cell, animal cell, fungal cell, or plant cell, etc.
  • the term “recombinant cell” refers to a cell (e.g., bacterial cell animal cell, fungal cell, or plant cell) that has been genetically modified from its native state. For instance, a recombinant cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into its DNA. These genetic modifications may be present in the chromosome of the cell, e.g., bacterial cell, plant cell, fungal cell, or animal cell.
  • a recombinant cell may have a genetic modification present on a plasmid in the cell or may comprise a heterologous nucleotide sequence(s) on a plasmid(s).
  • recombinant cells may comprise a heterologous nucleotide sequence(s) stably incorporated into its chromosome.
  • the term “recombinant bacterial cell” or “recombinant bacteria” refers to a bacterial cell or bacteria that have been genetically modified from their native state.
  • recombinant microorganism refers to a microorganism or a host cell that has been genetically modified from their native state.
  • a recombinant bacterial cell, microorganism, or host cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria, or bacterial cell, microorganism, or host cell or on a plasmid in the bacteria, or bacterial cell, microorganism, or host cell.
  • Recombinant bacterial cells, microorganisms, or host cells of the disclosure may comprise exogenous or heterologous nucleotide sequences on plasmids.
  • recombinant bacterial cells, microorganisms, or host cells may comprise exogenous or heterologous nucleotide sequences stably incorporated into their chromosome(s).
  • the term “gene” refers to a nucleic acid fragment that encodes a protein or fragment thereof, optionally including regulatory sequences preceding (5’ non-coding sequences) and following (3’ non-coding sequences) the coding sequence. In one embodiment, a “gene” does not include regulatory sequences preceding and following the coding sequence.
  • a “native gene” refers to a gene as found in nature, optionally with its own regulatory sequences preceding and following the coding sequence.
  • a “chimeric gene” refers to any gene that is not a native gene, optionally comprising regulatory sequences preceding and following the coding sequence, wherein the coding sequences and/or the regulatory sequences, in whole or in part, are not found together in nature.
  • a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory and coding sequences that are derived from the same source, but arranged differently than is found in nature.
  • the term “gene” is also meant to include a codon-optimized gene sequence, which is modified from a native gene sequence, e.g., to reflect the typical codon usage of the host organism, without altering the polypeptide encoded by a gene or nucleic acid molecule.
  • the term “gene” may also refer to a gene sequence which encodes a polypeptide that is not naturally occurring.
  • a gene may encode a polypeptide which is derived from a library of engineered, non-naturally occurring polypeptides.
  • the term “gene sequence” is meant to refer to a genetic sequence, e.g., a nucleic acid sequence.
  • the gene sequence or genetic sequence is meant to include a complete gene sequence or a partial gene sequence.
  • the gene sequence or genetic sequence is meant to include sequence that encodes a protein or polypeptide and is also meant to include genetic sequence that does not encode a protein or polypeptide, e.g., a regulatory sequence, leader sequence, signal sequence, or other non-protein coding sequence.
  • a heterologous gene or “heterologous sequence” refers to a nucleotide sequence that is not normally found in a given cell in nature.
  • a heterologous sequence encompasses a nucleic acid sequence that is exogenously introduced into a given cell.
  • Heterologous gene includes a native gene, or fragment thereof, that has been introduced into the host cell in a form that is different from the corresponding native gene.
  • a heterologous gene may include a native coding sequence that is a portion of a chimeric gene to include a native coding sequence that is a portion of a chimeric gene to include non-native regulatory regions that is reintroduced into the host cell.
  • a heterologous gene may also include a native gene, or fragment thereof, introduced into a non- native host cell.
  • a heterologous gene may also include a native gene, or fragment thereof, which has been edited within a host cell.
  • a heterologous gene may be foreign or native to the recipient cell; a nucleic acid sequence that is naturally found in a given cell but expresses an unnatural amount of the nucleic acid and/or the polypeptide which it encodes; and/or two or more nucleic acid sequences that are not found in the same relationship to each other in nature.
  • the term “endogenous gene” refers to a native gene in its natural location in the genome of an organism.
  • the term “transgene” refers to a gene that has been introduced into the host organism, e.g., host bacterial cell, genome.
  • bacteriostatic or “cytostatic” refers to a molecule or protein which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of recombinant bacterial cell of the disclosure.
  • bactericidal refers to a molecule or protein which is capable of killing the recombinant bacterial cell of the disclosure.
  • toxin refers to a protein, enzyme, or polypeptide fragment thereof, or other molecule which is capable of arresting, retarding, or inhibiting the growth, division, multiplication or replication of the recombinant bacterial cell of the disclosure, or which is capable of killing the recombinant bacterial cell of the disclosure.
  • the term “toxin” is intended to include bacteriostatic proteins and bactericidal proteins.
  • the term “toxin” is intended to include, but not limited to, lytic proteins, bacteriocins (e.g., microcins and colicins), gyrase inhibitors, polymerase inhibitors, transcription inhibitors, translation inhibitors, DNases, and RNases.
  • anti-toxin refers to a protein or enzyme which is capable of inhibiting the activity of a toxin.
  • anti-toxin is intended to include, but not limited to, immunity modulators, and inhibitors of toxin expression. Examples of toxins and antitoxins are known in the art and described in more detail infra.
  • coding region refers to a nucleotide sequence that codes for a specific amino acid sequence.
  • regulatory sequence refers to a nucleotide sequence located upstream (5’ non-coding sequences), within, or downstream (3’ non-coding sequences) of a coding sequence, and which influences the transcription, RNA processing, RNA stability, or translation of the associated coding sequence.
  • regulatory sequences include, but are not limited to, promoters, translation leader sequences, effector binding sites, and stem-loop structures.
  • the regulatory sequence comprises a promoter, e.g., an FNR responsive promoter.
  • “Operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a regulatory element is operably linked with a coding sequence when it is capable of affecting the expression of the gene coding sequence, regardless of the distance between the regulatory element and the coding sequence. More specifically, operably linked refers to a nucleic acid sequence, e.g., a gene encoding at least one uric acid catabolism enzyme, that is joined to a regulatory sequence in a manner which allows expression of the nucleic acid sequence, e.g., the gene(s) encoding the uric acid catabolism enzyme. In other words, the regulatory sequence acts in cis.
  • a gene may be “directly linked” to a regulatory sequence in a manner which allows expression of the gene.
  • a gene may be “indirectly linked” to a regulatory sequence in a manner which allows expression of the gene.
  • two or more genes may be directly or indirectly linked to a regulatory sequence in a manner which allows expression of the two or more genes.
  • a regulatory region or sequence is a nucleic acid that can direct transcription of a gene of interest and may comprise promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5′ and 3′ untranslated regions, transcriptional start sites, termination sequences, polyadenylation sequences, and introns.
  • a “promoter” as used herein refers to a nucleotide sequence that is capable of controlling the expression of a coding sequence or gene. Promoters are generally located 5’ of the sequence that they regulate. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from promoters found in nature, and/or comprise synthetic nucleotide segments. Those skilled in the art will readily ascertain that different promoters may regulate expression of a coding sequence or gene in response to a particular stimulus, e.g., in a cell- or tissue- specific manner, in response to different environmental or physiological conditions, or in response to specific compounds. Prokaryotic promoters are typically classified into two classes: inducible and constitutive.
  • an “inducible regulator region” refers to a regulatory region that is operably linked to one or more genes, wherein expression of the gene(s) is increased in the presence of an inducer of said regulatory region.
  • An “inducible promoter” refers to a promoter that initiates increased levels of transcription of the coding sequence or gene under its control in response to a stimulus or an exogenous environmental condition.
  • a “directly inducible promoter” refers to a regulatory region, wherein the regulatory region is operably linked to a gene encoding a protein or polypeptide, where, in the presence of an inducer of said regulatory region, the protein or polypeptide is expressed.
  • an “indirectly inducible promoter” refers to a regulatory system comprising two or more regulatory regions, for example, a first regulatory region that is operably linked to a first gene encoding a first protein, polypeptide, or factor, e.g., a transcriptional regulator, which is capable of regulating a second regulatory region that is operably linked to a second gene, the second regulatory region may be activated or repressed, thereby activating or repressing expression of the second gene.
  • inducible promoter Both a directly inducible promoter and an indirectly inducible promoter are encompassed by “inducible promoter.”
  • inducible promoters include, but are not limited to, an FNR promoter, a ParaC promoter, a ParaBAD promoter, a propionate promoter, and a PTetR promoter, each of which are described in more detail herein. Examples of other inducible promoters are provided herein below.
  • a “stably maintained” or “stable” host cell such as a bacterium
  • a host cell such as a bacterial host cell
  • non-native genetic material e.g., a uric acid degradation enzyme
  • the stable host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut.
  • the stable host cell such as a stable bacterium
  • copy number affects the stability of expression of the non-native genetic material.
  • copy number affects the level of expression of the non-native genetic material.
  • the term “expression” refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA derived from a nucleic acid, and/or to translation of an mRNA into a polypeptide
  • the term “plasmid” or “vector” refers to an extrachromosomal nucleic acid, e.g., DNA, construct that is not integrated into the genome of a host cell, such as a bacterial host cell. Plasmids are usually circular and capable of autonomous replication. Plasmids may be low-copy, medium-copy, or high-copy, as is well known in the art.
  • Plasmids may optionally comprise a selectable marker, such as an antibiotic resistance gene, which helps select for host cells, such as bacterial host cells, containing the plasmid and which ensures that the plasmid is retained in the host cell, such as a bacterial host cell.
  • a plasmid disclosed herein may comprise a nucleic acid sequence encoding a heterologous gene, e.g., a gene encoding at least one uric acid catabolism enzyme.
  • the term “transform” or “transformation” refers to the transfer of a nucleic acid fragment into a host cell, such as a host bacterial cell, resulting in genetically-stable inheritance.
  • Host cells such as host bacterial cells, comprising the transformed nucleic acid fragment are referred to as “recombinant” or “transgenic” or “transformed” cells or organisms.
  • one or more of the nucleic acid fragments may be retained in the cell, such as by integration into the genome of the cell, while the plasmid or vector itself may be removed from the cell.
  • the host cell is considered to be transformed with the nucleic acid fragments that were introduced into the cell regardless of whether the plasmid or vector is retained in the cell or not.
  • the term “genetic modification,” as used herein, refers to any genetic change.
  • Exemplary genetic modifications include those that increase, decrease, or abolish the expression of a gene, including, for example, modifications of native chromosomal or extrachromosomal genetic material. Exemplary genetic modifications also include the introduction of at least one plasmid, modification, mutation, base deletion, base addition, and/or codon modification of chromosomal or extrachromosomal genetic sequence(s), gene over-expression, gene amplification, gene suppression, promoter modification or substitution, gene addition (either single or multi-copy), antisense expression or suppression, or any other change to the genetic elements of a host cell, whether the change produces a change in phenotype or not.
  • Genetic modification can include the introduction of a plasmid, e.g., a plasmid comprising at least one uric acid catabolism enzyme operably linked to a promoter, into a host cell, such as a bacterial host cell. Genetic modification can also involve a targeted replacement in the chromosome, e.g., to replace a native gene promoter with an inducible promoter, regulated promoter, strong promoter, or constitutive promoter. Genetic modification can also involve gene amplification, e.g., introduction of at least one additional copy of a native gene into the chromosome of the cell. Alternatively, chromosomal genetic modification can involve a genetic mutation.
  • the term “genetic mutation” refers to a change or changes in a nucleotide sequence of a gene or related regulatory region that alters the nucleotide sequence as compared to its native or wild-type sequence. Mutations include, for example, substitutions, additions, and deletions, in whole or in part, within the wild-type sequence. Such substitutions, additions, or deletions can be single nucleotide changes (e.g., one or more point mutations), or can be two or more nucleotide changes, which may result in substantial changes to the sequence. Mutations can occur within the coding region of the gene as well as within the non-coding and regulatory sequence of the gene.
  • genetic mutation is intended to include silent and conservative mutations within a coding region as well as changes which alter the amino acid sequence of the polypeptide encoded by the gene.
  • a genetic mutation in a gene coding sequence may, for example, increase, decrease, or otherwise alter the activity (e.g., enzymatic activity) of the gene’s polypeptide product.
  • a genetic mutation in a regulatory sequence may increase, decrease, or otherwise alter the expression of sequences operably linked to the altered regulatory sequence.
  • Mutations include substitutions, insertions, deletions, and/or truncations of one or more specific amino acid residues or of one or more specific nucleotides or codons in the polypeptide or polynucleotide.
  • Mutagenesis and directed evolution methods are well known in the art for creating variants. See, e.g., U.S. Pat. No.7,783,428; U.S. Pat. No.6,586,182; U.S. Pat. No.6,117,679; and Ling, et al., 1999, "Approaches to DNA mutagenesis: an overview," Anal. Biochem., 254(2):157-78; Smith, 1985, “In vitro mutagenesis,” Ann. Rev.
  • the term "inactivated” as applied to a gene refers to any genetic modification that decreases or eliminates the expression of the gene and/or the functional activity of the corresponding gene product (mRNA and/or protein).
  • inactivated encompasses complete or partial inactivation, suppression, deletion, interruption, blockage, promoter alterations, antisense RNA, dsRNA, or down-regulation of a gene. This can be accomplished, for example, by gene "knockout,” inactivation, mutation (e.g., insertion, deletion, point, or frameshift mutations that disrupt the expression or activity of the gene product), or by use of inhibitory RNAs (e.g., sense, antisense, or RNAi technology).
  • a deletion may encompass all or part of a gene's coding sequence.
  • knockout refers to the deletion of most (at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) or all (100%) of the coding sequence of a gene. In some embodiments, any number of nucleotides can be deleted, from a single base to an entire piece of a chromosome.
  • Exogenous environmental condition(s)” or “environmental conditions” refer to settings or circumstances under which the promoter described herein is directly or indirectly induced. The phrase is meant to refer to the environmental conditions external to the engineered microorganism, but endogenous or native to the host subject environment.
  • exogenous and endogenous may be used interchangeably to refer to environmental conditions in which the environmental conditions are endogenous to a mammalian body, but external or exogenous to an intact microorganism cell.
  • the exogenous environmental conditions are specific to the gut of a mammal.
  • the exogenous environmental conditions are specific to the upper gastrointestinal tract of a mammal.
  • the exogenous environmental conditions are specific to the lower gastrointestinal tract of a mammal.
  • the exogenous environmental conditions are specific to the small intestine of a mammal.
  • the exogenous environmental conditions are low-oxygen, microaerobic, or anaerobic conditions, such as the environment of the mammalian gut.
  • exogenous environmental conditions refer to the presence of molecules or metabolites that are specific to the mammalian gut in a healthy or disease-state.
  • the exogenous environmental condition is a tissue-specific or disease-specific metabolite or molecule(s).
  • the exogenous environmental condition is a low-pH environment.
  • the genetically engineered microorganism of the disclosure comprises a pH-dependent promoter.
  • the genetically engineered microorganism of the disclosure comprises an oxygen level-dependent promoter.
  • bacteria have evolved transcription factors that are capable of sensing oxygen levels. Different signaling pathways may be triggered by different oxygen levels and occur with different kinetics.
  • exogenous environmental conditions or “environmental conditions” also refers to settings or circumstances or environmental conditions external to the engineered microorganism, which relate to in vitro culture conditions of the microorganism. “Exogenous environmental conditions” may also refer to the conditions during growth, production, and manufacture of the organism. Such conditions include aerobic culture conditions, anaerobic culture conditions, low oxygen culture conditions and other conditions under set oxygen concentrations.
  • Such conditions also include the presence of a chemical and/or nutritional inducer, such as tetracycline, arabinose, IPTG, rhamnose, and the like in the culture medium.
  • a chemical and/or nutritional inducer such as tetracycline, arabinose, IPTG, rhamnose, and the like in the culture medium.
  • Such conditions also include the temperatures at which the microorganisms are grown prior to in vivo administration. For example, using certain promoter systems, certain temperatures are permissive to expression of a payload, while other temperatures are non-permissive. Oxygen levels, temperature and media composition influence such exogenous environmental conditions.
  • Such conditions affect proliferation rate, rate of induction of the payload or gene of interest, e.g., uric acid catabolism gene, other regulators, and overall viability and metabolic activity of the strain during strain production.
  • the exogenous environmental condition(s) and/or signal(s) stimulates the activity of an inducible promoter.
  • the exogenous environmental condition(s) and/or signal(s) that serves to activate the inducible promoter is not naturally present within the gut of a mammal.
  • the inducible promoter is stimulated by a molecule or metabolite that is administered in combination with the pharmaceutical composition of the disclosure, for example, tetracycline, arabinose, or any biological molecule that serves to activate an inducible promoter.
  • the exogenous environmental condition(s) and/or signal(s) is added to culture media comprising a recombinant bacterial cell of the disclosure.
  • the exogenous environmental condition that serves to activate the inducible promoter is naturally present within the gut of a mammal (for example, low oxygen or anaerobic conditions, or biological molecules involved in an inflammatory response).
  • the loss of exposure to an exogenous environmental condition inhibits the activity of an inducible promoter, as the exogenous environmental condition is not present to induce the promoter (for example, an aerobic environment outside the gut).
  • oxygen level-dependent promoter or “oxygen level-dependent regulatory region” refers to a nucleic acid sequence to which one or more oxygen level-sensing transcription factors is capable of binding, wherein the binding and/or activation of the corresponding transcription factor activates downstream gene expression.
  • oxygen level-dependent transcription factors include, but are not limited to, FNR, ANR, and DNR.
  • FNR-responsive promoters Corresponding FNR-responsive promoters, ANR-responsive promoters, and DNR-responsive promoters are known in the art (see, e.g., Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991; Hasegawa et al., 1998; Hoeren et al., 1993; Salmon et al., 2003).
  • Non- limiting examples are shown in Table 1.
  • a promoter (PfnrS) was derived from the E.
  • coli Nissle fumarate and nitrate reductase gene S that is known to be highly expressed under conditions of low or no environmental oxygen (Durand and Storz, 2010; Boysen et al, 2010).
  • the PfnrS promoter is activated under anaerobic and/or low oxygen conditions by the global transcriptional regulator FNR that is naturally found in Nissle. Under anaerobic and/or low oxygen conditions, FNR forms a dimer and binds to specific sequences in the promoters of specific genes under its control, thereby activating their expression. However, under aerobic conditions, oxygen reacts with iron-sulfur clusters in FNR dimers and converts them to an inactive form.
  • PfnrS inducible promoter is adopted to modulate the expression of proteins or RNA.
  • PfnrS is used interchangeably in this application as FNRS, fnrS, FNR, P-FNRS promoter and other such related designations to indicate the promoter PfnrS. Table 1.
  • a “non-native” nucleic acid sequence refers to a nucleic acid sequence not normally present in a host cell, such as a bacterium, e.g., an extra copy of an endogenous sequence, or a heterologous sequence such as a sequence from a different species, strain, or substrain of bacteria, or a sequence that is modified and/or mutated as compared to the unmodified sequence from bacteria of the same subtype.
  • the non-native nucleic acid sequence is a synthetic, non- naturally occurring sequence (see, e.g., Purcell et al., 2013).
  • the non-native nucleic acid sequence may be a regulatory region, a promoter, a gene, and/or one or more genes in a gene cassette.
  • “non-native” refers to two or more nucleic acid sequences that are not found in the same relationship to each other in nature.
  • the non-native nucleic acid sequence may be present on a plasmid or chromosome.
  • multiple copies of any regulatory region, promoter, gene, and/or gene cassette may be present in the host cell, such as a bacterium, wherein one or more copies of the regulatory region, promoter, gene, and/or gene cassette may be mutated or otherwise altered as described herein.
  • the genetically engineered host cell such as a bacteria
  • the genetically engineered host cell, such as a bacteria, of the invention comprise a gene encoding a phenylalanine-metabolizing enzyme that is operably linked to a directly or indirectly inducible promoter that is not associated with said gene in nature, e.g., an FNR promoter operably linked to a gene encoding a uric acid catabolism gene.
  • Constant promoter refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and/or to which it is operably linked.
  • Constitutive promoters and variants are well known in the art and include, but are not limited to, BBa_J23100, a constitutive Escherichia coli ⁇ S promoter (e.g., an osmY promoter (International Genetically Engineered Machine (iGEM) Registry of Standard Biological Parts Name BBa_J45992; BBa_J45993)), a constitutive Escherichia coli ⁇ 32 promoter (e.g., htpG heat shock promoter (BBa_J45504)), a constitutive Escherichia coli ⁇ 70 promoter (e.g., lacq promoter (BBa_J54200; BBa_J56015), E.
  • a constitutive Escherichia coli ⁇ S promoter e.g., an
  • coli CreABCD phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa_K119000; BBa_K119001); M13K07 gene I promoter (BBa_M13101); M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter (BBa_M13108), M13110 (BBa_M13110)), a constitutive Bacillus subtilis ⁇ A promoter (e.g., promoter veg (BBa_K143013), promoter 43 (BBa_K143013), P liaG (BBa_K823000), P lepA (BBa_K823002)
  • “Gut” refers to the organs, glands, tracts, and systems that are responsible for the transfer and digestion of food, absorption of nutrients, and excretion of waste.
  • the gut comprises the gastrointestinal (GI) tract, which starts at the mouth and ends at the anus, and additionally comprises the esophagus, stomach, small intestine, and large intestine.
  • the gut also comprises accessory organs and glands, such as the spleen, liver, gallbladder, and pancreas.
  • the upper gastrointestinal tract comprises the esophagus, stomach, and duodenum of the small intestine.
  • the lower gastrointestinal tract comprises the remainder of the small intestine, i.e., the jejunum and ileum, and all of the large intestine, i.e., the cecum, colon, rectum, and anal canal.
  • Bacteria can be found throughout the gut, e.g., in the gastrointestinal tract, and particularly in the intestines.
  • the genetically engineered bacteria are active in the gut.
  • the genetically engineered bacteria are active in the large intestine.
  • the genetically engineered bacteria are active in the small intestine.
  • the genetically engineered bacteria are active in the small intestine and in the large intestine.
  • the genetically engineered bacteria transit through the small intestine. In some embodiments, the genetically engineered bacteria have increased residence time in the small intestine. In some embodiments, the genetically engineered bacteria colonize the small intestine. In some embodiments, the genetically engineered bacteria do not colonize the small intestine. In some embodiments, the genetically engineered bacteria have increased residence time in the gut. In some embodiments, the genetically engineered bacteria colonize the small intestine. In some embodiments, the genetically engineered bacteria do not colonize the gut.
  • the term “low oxygen” is meant to refer to a level, amount, or concentration of oxygen (O2) that is lower than the level, amount, or concentration of oxygen that is present in the atmosphere (e.g., ⁇ 21% O2; ⁇ 160 torr O2)).
  • the term “low oxygen condition or conditions” or “low oxygen environment” refers to conditions or environments containing lower levels of oxygen than are present in the atmosphere.
  • the term “low oxygen” is meant to refer to the level, amount, or concentration of oxygen (O 2 ) found in a mammalian gut, e.g., lumen, stomach, small intestine, duodenum, jejunum, ileum, large intestine, cecum, colon, distal sigmoid colon, rectum, and anal canal.
  • O 2 oxygen
  • the term “low oxygen” is meant to refer to a level, amount, or concentration of O 2 that is 0-60 mmHg O 2 (0-60 torr O 2) (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 mmHg O 2 ), including any and all incremental fraction(s) thereof (e.g., 0.2 mmHg, 0.5 mmHg O 2 , 0.75 mmHg O 2 , 1.25 mmHg O 2 , 2.175 mmHg O 2 , 3.45 mmHg O 2 , 3.75 mmHg O 2 , 4.5 mmHg O 2 , 6.8 mmHg O 2
  • “low oxygen” refers to about 60 mmHg O 2 or less (e.g., 0 to about 60 mmHg O 2) .
  • the term “low oxygen” may also refer to a range of O 2 levels, amounts, or concentrations between 0-60 mmHg O 2 (inclusive), e.g., 0-5 mmHg O 2 , ⁇ 1.5 mmHg O 2 , 6-10 mmHg, ⁇ 8 mmHg, 47-60 mmHg, etc. which listed exemplary ranges are listed here for illustrative purposes and not meant to be limiting in any way.
  • the term “low oxygen” is meant to refer to the level, amount, or concentration of oxygen (O2) found in a mammalian organ or tissue other than the gut, e.g., urogenital tract, tumor tissue, etc. in which oxygen is present at a reduced level, e.g., at a hypoxic or anoxic level.
  • “low oxygen” is meant to refer to the level, amount, or concentration of oxygen (O2) present in partially aerobic, semi aerobic, microaerobic, nanoaerobic, microoxic, hypoxic, anoxic, and/or anaerobic conditions.
  • Table 2 summarizes the amount of oxygen present in various organs and tissues.
  • DO dissolved oxygen
  • the term “low oxygen” is meant to refer to a level, amount, or concentration of oxygen (O2) that is about 6.0 mg/L DO or less, e.g., 6.0 mg/L, 5.0 mg/L, 4.0 mg/L, 3.0 mg/L, 2.0 mg/L, 1.0 mg/L, or 0 mg/L, and any fraction therein, e.g., 3.25 mg/L, 2.5 mg/L, 1.75 mg/L, 1.5 mg/L, 1.25 mg/L, 0.9 mg/L, 0.8 mg/L, 0.7 mg/L, 0.6 mg/L, 0.5 mg/L, 0.4 mg/L, 0.3 mg/L, 0.2 mg/L and 0.1 mg/L DO, which exemplary fractions are listed here for illustrative purposes and not meant to be limiting in any way.
  • the level of oxygen in a liquid or solution may also be reported as a percentage of air saturation or as a percentage of oxygen saturation (the ratio of the concentration of dissolved oxygen (O2) in the solution to the maximum amount of oxygen that will dissolve in the solution at a certain temperature, pressure, and salinity under stable equilibrium).
  • Well-aerated solutions e.g., solutions subjected to mixing and/or stirring
  • oxygen producers or consumers are 100% air saturated.
  • the term “low oxygen” is meant to refer to 40% air saturation or less, e.g., 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, and 0% air saturation, including any and all incremental fraction(s) thereof (e.g., 30.25%, 22.70%, 15.5%, 7.7%, 5.0%, 2.8%, 2.0%, 1.65%, 1.0%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%.0.44%, 0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%.
  • any range of air saturation levels between 0-40%, inclusive e.g., 0- 5%, 0.05 – 0.1%, 0.1-0.2%, 0.1-0.5%, 0.5 – 2.0%, 0-10%, 5-10%, 10-15%, 15-20%, 20-25%, 25- 30%, etc.
  • the exemplary fractions and ranges listed here are for illustrative purposes and not meant to be limiting in any way.
  • the term “low oxygen” is meant to refer to 9% O 2 saturation or less, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0%, O 2 saturation, including any and all incremental fraction(s) thereof (e.g., 6.5%, 5.0%, 2.2%, 1.7%, 1.4%, 0.9%, 0.8%, 0.75%, 0.68%, 0.5%.0.44%, 0.3%, 0.25%, 0.2%, 0.1%, 0.08%, 0.075%, 0.058%, 0.04%.0.032%, 0.025%, 0.01%, etc.) and any range of O2 saturation levels between 0-9%, inclusive (e.g., 0-5%, 0.05 – 0.1%, 0.1- 0.2%, 0.1-0.5%, 0.5 – 2.0%, 0-8%, 5-7%, 0.3-4.2% O2, etc.).
  • Microorganism refers to an organism or microbe of microscopic, submicroscopic, or ultramicroscopic size that typically consists of a single cell. Examples of microorganisms include bacteria, yeast, viruses, parasites, fungi, certain algae, and protozoa.
  • the microorganism is engineered (“genetically engineered microorganism”) to produce one or more therapeutic molecules or proteins of interest.
  • the microorganism is engineered to take up and catabolize certain metabolites or other compounds from its environment, e.g., the gut.
  • the microorganism is engineered to synthesize certain beneficial metabolites or other compounds (synthetic or naturally occurring) and release them into its environment.
  • the engineered microorganism is an engineered bacterium.
  • the engineered microorganism is an engineered virus.
  • the engineered microorganism is an engineered fungus.
  • the engineered microorganism is an engineered algae.
  • the engineered microorganism is an engineered yeast.
  • the engineered microorganism is an engineered protozoa.
  • the engineered microorganism is an engineered parasite.
  • “Host cell” refers to a cell that can be used to express a polynucleotide, such as a polynucleotide that encodes a uric acid catabolism enzyme, such as a uricase, and/or a urate importer.
  • “Non-pathogenic bacteria” refer to bacteria that are not capable of causing disease or harmful responses in a host. In some embodiments, non-pathogenic bacteria are Gram-negative bacteria. In some embodiments, non-pathogenic bacteria are Gram-positive bacteria. In some embodiments, non-pathogenic bacteria are commensal bacteria, which are present in the indigenous microbiota of the gut.
  • non-pathogenic bacteria examples include, but are not limited to, Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Clostridium, Enterococcus, Escherichia, Lactobacillus, Lactococcus, Saccharomyces, and Staphylococcus, e.g., Bacillus coagulans, Bacillus subtilis, Bacteroides fragilis, Bacteroides subtilis, Bacteroides thetaiotaomicron, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Clostridium butyricum, Enterococcus faecium, Escherichia coli, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus
  • Naturally pathogenic bacteria may be genetically engineered to provide reduce or eliminate pathogenicity.
  • “Probiotic” is used to refer to live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an appropriate amount of the microorganism.
  • the host organism is a mammal.
  • the host organism is a human.
  • Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic.
  • probiotic bacteria examples include, but are not limited to, Bifidobacteria, Escherichia, Lactobacillus, and Saccharomyces, e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, and Saccharomyces boulardii (Dinleyici et al., 2014; U.S. Patent No.5,589,168; U.S. Patent No.6,203,797; U.S. Patent 6,835,376).
  • Bifidobacterium bifidum Enterococcus faecium
  • Escherichia coli Escherichia coli strain Nissle
  • Lactobacillus acidophilus Lactobacillus bulg
  • the probiotic may be a variant or a mutant strain of bacterium (Arthur et al., 2012; Cuevas-Ramos et al., 2010; Olier et al., 2012; Nougayrede et al., 2006).
  • Non-pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability.
  • Non-pathogenic bacteria may be genetically engineered to provide probiotic properties.
  • Probiotic bacteria may be genetically engineered to enhance or improve probiotic properties.
  • the term “auxotroph” or “auxotrophic” refers to an organism that requires a specific factor, e.g., an amino acid, a sugar, or other nutrient, to support its growth.
  • an “auxotrophic modification” is a genetic modification that causes the organism to die in the absence of an exogenously added nutrient essential for survival or growth because it is unable to produce said nutrient.
  • essential gene refers to a gene which is necessary to for cell growth and/or survival. Essential genes are described in more detail infra and include, but are not limited to, DNA synthesis genes (such as thyA), cell wall synthesis genes (such as dapA), and amino acid genes (such as serA and metA).
  • the terms “modulate” and “treat” and their cognates refer to an amelioration of a disease, disorder, and/or condition, or at least one discernible symptom thereof.
  • module and “treat” refer to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient.
  • modulate” and “treat” refer to inhibiting the progression of a disease, disorder, and/or condition, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both.
  • modulate” and “treat” refer to slowing the progression or reversing the progression of a disease, disorder, and/or condition.
  • prevent and its cognates refer to delaying the onset or reducing the risk of acquiring a given disease, disorder and/or condition or a symptom associated with such disease, disorder, and/or condition.
  • Those in need of treatment may include individuals already having a particular medical disease, as well as those at risk of having, or who may ultimately acquire the disease.
  • the need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disease, the presence or progression of a disease, or likely receptiveness to treatment of a subject having the disease.
  • Disorders associated with or involved with uric acid e.g., hyperuricemia, may be caused by inborn genetic mutations for which there are no known cures.
  • Diseases can also be secondary to other conditions, e.g., an intestinal disorder or a bacterial infection. Treating diseases associated with uric acid degradation may encompass reducing normal levels of uric acid, reducing excess levels of uric acid, or eliminating uric acid, and does not necessarily encompass the elimination of the underlying disease.
  • the terms “disease or disorder associated with uric acid,” “disease associated with uric acid degradation” or a “disorder associated with uric acid degradation” is a disease or disorder involving the abnormal, e.g., increased, levels of uric acid in a subject.
  • a disease or disorder associated with uric acid is hyperuricemia.
  • a disease or disorder associated with uric acid is gout.
  • amino acid refers to a class of organic compounds that contain at least one amino group and one carboxyl group.
  • Amino acids include leucine, isoleucine, valine, arginine, lysine, asparagine, serine, glycine, glutamine, tryptophan, methionine, threonine, cysteine, tyrosine, phenylalanine, glutamic acid, aspartic acid, alanine, histidine, and proline.
  • uric acid degradation or “uric acid catabolism” refers to the processing, breakdown and/or degradation of uric acid into other compounds that are not associated with the disease associated with uric acid, such as hyperuricemia and/or gout, or other compounds which can be utilized by the bacterial cell.
  • uric acid degrading enzyme or “uric acid degradation enzyme” refers to the processing, breakdown, and/or degradation of uric acid.
  • a uric acid degradation enzyme refers to the processing, breakdown, and/or degradation of uric acid into, for example, hydroxyisourate and/or allantoin.
  • a “uric acid degrading enzyme” or “uric acid degradation enzyme” may refer to an enzyme which works upstream to degrade a precursor of uric acid, thereby decreasing downstream levels of uric acid.
  • a uric acid degradation enzyme degrades guanosine.
  • a uric acid degradation enzyme degrades adenosine.
  • payload refers to one or more molecules of interest to be produced by a genetically engineered microorganism, such as a bacterium or a virus.
  • the payload is a therapeutic payload, e.g., a uric acid degradation enzyme or a transporter polypeptide.
  • the payload is a regulatory molecule, e.g., a transcriptional regulator such as FNR.
  • the payload comprises a regulatory element, such as a promoter or a repressor.
  • the payload comprises an inducible promoter, such as from FNRS.
  • the payload comprises a repressor element, such as a kill switch.
  • the payload is encoded by a gene or multiple genes or an operon.
  • the payload is produced by a biosynthetic or biochemical pathway, wherein the biosynthetic or biochemical pathway may optionally be endogenous to the microorganism.
  • the genetically engineered microorganism comprises two or more payloads.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20.
  • therapeutically effective dose and “therapeutically effective amount” are used to refer to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a condition.
  • a therapeutically effective amount may, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms of a disease or condition associated with excess uric acid levels.
  • a therapeutically effective amount, as well as a therapeutically effective frequency of administration, can be determined by methods known in the art and discussed below.
  • polypeptide includes “polypeptide” as well as “polypeptides,” and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (i.e., peptide bonds).
  • polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
  • peptides “dipeptides,” “tripeptides, “oligopeptides,” “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms.
  • dipeptide refers to a peptide of two linked amino acids.
  • tripeptide refers to a peptide of three linked amino acids.
  • polypeptide is also intended to refer to the products of post- expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, or modification by non-naturally occurring amino acids.
  • a polypeptide may be derived from a natural biological source or produced by recombinant technology.
  • a polypeptide may be a naturally occurring polypeptide or alternatively may be a polypeptide not naturally occurring, such as a polypeptide identified from a library of engineered polypeptides.
  • the polypeptide is produced by the cell, e.g., genetically engineered or recombinant bacteria, fungus, parasite, plant, animal, yeast or virus, of the current disclosure.
  • a polypeptide of the invention may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids.
  • Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure.
  • polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides, which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, are referred to as unfolded.
  • the term “peptide” or “polypeptide” may refer to an amino acid sequence that corresponds to a protein or a portion of a protein or may refer to an amino acid sequence that corresponds with non- protein sequence, e.g., a sequence selected from a regulatory peptide sequence, leader peptide sequence, signal peptide sequence, linker peptide sequence, and other peptide sequence.
  • an “isolated” polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required.
  • Recombinantly produced polypeptides and proteins expressed in host cells including but not limited to bacterial or mammalian cells, are considered isolated for purposed of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.
  • Recombinant peptides, polypeptides or proteins refer to peptides, polypeptides or proteins produced by recombinant DNA techniques, i.e.
  • fragments of polypeptides of the present invention include proteolytic fragments, as well as deletion fragments.
  • Fragments also include specific antibody or bioactive fragments or immunologically active fragments derived from any polypeptides described herein. Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants may be produced using mutagenesis methods known in the art. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. [0153] Polypeptides also include fusion proteins. As used herein, the term “variant” includes a fusion protein, which comprises a sequence of the original peptide or sufficiently similar to the original peptide. As used herein, the term “fusion protein” refers to a chimeric protein comprising amino acid sequences of two or more different proteins.
  • Fusion proteins result from well known in vitro recombination techniques. Fusion proteins may have a similar structural function (but not necessarily to the same extent), and/or similar regulatory function (but not necessarily to the same extent), and/or similar biochemical function (but not necessarily to the same extent) and/or immunological activity (but not necessarily to the same extent) as the individual original proteins which are the components of the fusion proteins. “Derivatives” include but are not limited to peptides, which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. “Similarity” between two peptides is determined by comparing the amino acid sequence of one peptide to the sequence of a second peptide.
  • amino acid of one peptide is similar to the corresponding amino acid of a second peptide if it is identical or a conservative amino acid substitution.
  • Conservative substitutions include those described in Dayhoff, M. O., ed., The Atlas of Protein Sequence and Structure 5, National Biomedical Research Foundation, Washington, D.C. (1978), and in Argos, EMBO J.8 (1989), 779-785.
  • amino acids belonging to one of the following groups represent conservative changes or substitutions: Ala, Pro, Gly, Gln, Asn, Ser, Thr, Cys, Ser, Tyr, Thr, Val, Ile, Leu, Met, Ala, Phe, Lys, Arg, His, Phe, Tyr, Trp, His, Asp, and Glu.
  • the term “sufficiently similar” means a first amino acid sequence that contains a sufficient or minimum number of identical or equivalent amino acid residues relative to a second amino acid sequence such that the first and second amino acid sequences have a common structural domain and/or common functional activity.
  • amino acid sequences that comprise a common structural domain that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, identical are defined herein as sufficiently similar.
  • variants will be sufficiently similar to the amino acid sequence of the peptides of the invention. Such variants generally retain the functional activity of the peptides of the present invention.
  • Variants include peptides that differ in amino acid sequence from the native and wild-type peptide, respectively, by way of one or more amino acid deletion(s), addition(s), and/or substitution(s). These may be naturally occurring variants as well as artificially designed ones.
  • the term “percent identity” refers to a quantitative measurement of the similarity between two sequences (e.g., nucleic acid or amino acid sequence). In some embodiments, the “percent identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993.
  • a first nucleic acid sequence may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least about 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of a second nucleic acid.
  • a first polypeptide may comprise an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least about 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the amino acid sequence of a second polypeptide.
  • linker refers to synthetic or non-native or non-naturally-occurring amino acid sequences that connect or link two polypeptide sequences, e.g., that link two polypeptide domains.
  • the term “synthetic” refers to amino acid sequences that are not naturally occurring. Exemplary linkers are described herein. Additional exemplary linkers are provided in US 20140079701, the contents of which are herein incorporated by reference in its entirety. [0157]
  • the term “codon-optimized sequence” refers to a sequence, which was modified from an existing coding sequence, or designed, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host organism.
  • codon-optimized refers to the modification of codons in the gene or coding regions of a nucleic acid molecule to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the nucleic acid molecule. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of the host organism.
  • a “codon-optimized sequence” refers to a sequence, which was modified from an existing coding sequence, or designed, for example, to improve translation in an expression host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence.
  • the improvement of transcription and/or translation involves increasing the level of transcription and/or translation. In some embodiments, the improvement of transcription and/or translation involves decreasing the level of transcription and/or translation.
  • codon optimization is used to fine-tune the levels of expression from a construct of interest. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host organism. Many organisms display a bias or preference for use of particular codons to code for insertion of a particular amino acid in a growing polypeptide chain. Codon preference or codon bias, differences in codon usage between organisms, is allowed by the degeneracy of the genetic code, and is well documented among many organisms.
  • Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent, inter alia, on the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • the predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.
  • the term “transporter” or “importer” is meant to refer to a mechanism, e.g., protein or proteins, for importing a molecule, e.g., urate or uric acid, toxin, metabolite, substrate, etc. into a host cell from the extracellular milieu.
  • a uric acid transporter such as UacT imports uric acid into a host cell, e.g., microorganism, e.g., bacteria.
  • uric acid transporter and “urate transporter” are used interchangeably herein.
  • phage and “bacteriophage” are used interchangeably herein.
  • phage or bacteriophage
  • prophage refers to the genomic material of a bacteriophage, which is integrated into a replicon of the host cell and replicates along with the host. The prophage may be able to produce phages if specifically activated.
  • prophage is not able to produce phages or has never done so (i.e., defective or cryptic prophages). In some cases, prophage also refers to satellite phages.
  • prophage and “endogenous phage” are used interchangeably herein. “Endogenous phage” or “endogenous prophage” also refers to a phage that is present in the natural state of a bacterium (and its parental strain).
  • phage knockout” or “inactivated phage” refers to a phage which has been modified so that it can either no longer produce and/or package phage particles or it produces fewer phage particles than the wild type phage sequence.
  • the inactivated phage or phage knockout refers to the inactivation of a temperate phage in its lysogenic state, i.e., to a prophage.
  • a modification refers to a mutation in the phage; such mutations include insertions, deletions (partial or complete deletion of phage genome), substitutions, inversions, at one or more positions within the phage genome, e.g., within one or more genes within the phage genome.
  • phage-free”, “phage free” and “phageless” are used interchangeably to characterize a bacterium or strain which contains one or more prophages, one or more of which have been modified.
  • the modification can result in a loss of the ability of the prophage to be induced or release phage particles.
  • the modification can result in less efficient or less frequent induction or less efficient or less frequent phage release as compared to the isogenic strain without the modification.
  • Ability to induce and release phage can be measured using a plaque assay as described herein.
  • phage induction refers to the part of the life cycle of a lysogenic prophage, in which the lytic phage genes are activated, phage particles are produced and lysis occurs.
  • a "pharmaceutical composition” refers to a preparation of bacterial cells disclosed herein with other components such as a physiologically suitable carrier and/or excipient.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be used interchangeably refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered bacterial compound. An adjuvant is included under these phrases.
  • pharmaceutically acceptable carrier and “pharmaceutically acceptable carrier” which may be used interchangeably refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered bacterial compound. An adjuvant is included under these phrases.
  • the articles “a” and “an,” as used herein, should be understood to mean “at least one,” unless clearly indicated to the contrary.
  • a heterologous gene encoding a uric acid degradation enzyme should be understood to mean “at least one heterologous gene encoding at least one uric acid degradation enzyme.”
  • a heterologous gene encoding a uric acid transporter should be understood to mean “at least one heterologous gene encoding at least one uric acid transporter.”
  • A, B, and/or C indicates that the selection may be A alone; B alone; C alone; A and B; A and C; B and C; or A, B, and C.
  • the phrase “and/or” may be used interchangeably with “at least one of” or “one or more of” the elements in a list.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub- range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • Any suitable host cell may be used to express any of the enzymes disclosed herein, such as uric acid catabolism enzymes (e.g., uricases) and urate importers.
  • Suitable host cells include, but are not limited to, bacterial cells (e.g., E.
  • yeast host cells include, but are not limited to: Candida, Hansenula, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, and Yarrowia.
  • the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccaromyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, or Yarrowia lipolytica.
  • the yeast strain is an industrial polyploid yeast strain.
  • Other non- limiting examples of fungal cells include cells obtained from Aspergillus spp., Penicillium spp., Fusarium spp., Rhizopus spp., Acremonium spp., Neurospora spp., Sordaria spp., Magnaporthe spp., Allomyces spp., Ustilago spp., Botrytis spp., and Trichoderma spp.
  • the host cell is an algal cell such as Chlamydomonas (e.g., C. Reinhardtii) and Phormidium (P.
  • the host cell is an animal cell.
  • the host cell is a mammalian cell, including, for example, a human cell (e.g., 293, HeLa, WI38, PER.C6 or Bowes melanoma cells), a mouse cell (e.g., 3T3, NS0, NS1 or Sp2/0), a hamster cell (e.g., CHO or BHK), or a monkey cell (e.g., COS, FRhL or Vero).
  • the cell is a hybridoma cell line.
  • the host cell is a bacterial cell.
  • the disclosure provides a cell, e.g., a bacterial cell, a yeast cell, a fungal cell, etc., that comprises a gene, e.g., a heterologous gene, encoding a uric acid degradation, e.g., uricase, enzyme.
  • the cell is a bacterial cell, e.g., a non-pathogenic bacterial cell.
  • the bacterial cell is a commensal bacterial cell.
  • the bacterial cell is a probiotic bacterial cell.
  • the bacterial cell is selected from the group consisting of a Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides subtilis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Clostridium butyricum, Clostridium scindens, Escherichia coli, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus reuteri, Lactococcus lactis, and Oxalobacter formigenes bacterial cell.
  • a Bacteroides fragilis Bacteroides thetaiotaomicron
  • Bacteroides subtilis Bacteroides subtilis
  • Bifidobacterium animalis Bifidobacterium bifidum
  • Bifidobacterium infantis Bifidobacterium lactis
  • the bacterial cell is a Bacteroides fragilis bacterial cell. In one embodiment, the bacterial cell is a Bacteroides thetaiotaomicron bacterial cell. In one embodiment, the bacterial cell is a Bacteroides subtilis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium animalis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium bifidum bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium infantis bacterial cell. In one embodiment, the bacterial cell is a Bifidobacterium lactis bacterial cell.
  • the bacterial cell is a Clostridium butyricum bacterial cell. In one embodiment, the bacterial cell is a Clostridium scindens bacterial cell. In one embodiment, the bacterial cell is an Escherichia coli bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus acidophilus bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus plantarum bacterial cell. In one embodiment, the bacterial cell is a Lactobacillus reuteri bacterial cell. In one embodiment, the bacterial cell is a Lactococcus lactis bacterial cell. In one embodiment, the bacterial cell is a Oxalobacter formigenes bacterial cell.
  • the bacterial cell does not include Oxalobacter formigenes.
  • the bacterial cell is a Gram-positive bacterial cell.
  • the bacterial cell is a Gram-negative bacterial cell.
  • the bacterial cell is Escherichia coli strain Nissle 1917 (E. coli Nissle), a Gram-positive bacterium of the Enterobacteriaceae family that “has evolved into one of the best characterized probiotics” (Ukena et al., 2007). The strain is characterized by its “complete harmlessness” (Schultz, 2008), and “has GRAS (generally recognized as safe) status” (Reister et al., 2014, emphasis added).
  • E. coli Nissle “lacks prominent virulence factors (e.g., E. coli ⁇ -hemolysin, P-fimbrial adhesins)” (Schultz, 2008), and E. coli Nissle “does not carry pathogenic adhesion factors and does not produce any enterotoxins or cytotoxins, it is not invasive, not uropathogenic” (Sonnenborn et al., 2009). As early as in 1917, E. coli Nissle was packaged into medicinal capsules, called Mutaflor, for therapeutic use. E.
  • coli Nissle has since been used to treat ulcerative colitis in humans in vivo (Rembacken et al., 1999), to treat inflammatory bowel disease, Crohn’s disease, and pouchitis in humans in vivo (Schultz, 2008), and to inhibit enteroinvasive Salmonella, Legionella, Yersinia, and Shigella in vitro (Altenhoefer et al., 2004). It is commonly accepted that E. coli Nissle’s “therapeutic efficacy and safety have convincingly been proven” (Ukena et al., 2007). [0174] In one embodiment, the recombinant bacterial cell does not colonize the subject.
  • the bacterial cell is a genetically engineered bacterial cell.
  • the bacterial cell is a recombinant bacterial cell.
  • the disclosure comprises a colony of bacterial cells.
  • the disclosure provides a recombinant bacterial culture which comprises bacterial cells disclosed herein.
  • the disclosure provides a recombinant bacterial culture which reduces levels of uric acid in the media of the culture.
  • the levels of uric acid are reduced by about 50%, about 75%, or about 100% in the media of the cell culture.
  • the levels of uric acid are reduced by about two-fold, three-fold, four-fold, five-fold, six- fold, seven-fold, eight-fold, nine-fold, or ten-fold in the media of the cell culture.
  • the levels of uric acid are reduced below the limit of detection in the media of the cell culture.
  • the gene encoding a uric acid degradation enzyme is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions.
  • the gene encoding a uric acid degradation enzyme is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low- oxygen or anaerobic conditions.
  • the genetically engineered bacterium comprising a uric acid degradation enzyme is an auxotroph.
  • the genetically engineered bacterium is an auxotroph selected from a cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thi1 auxotroph.
  • the engineered bacteria have more than one auxotrophy, for example, they may be a ⁇ thyA and ⁇ dapA auxotroph.
  • the genetically engineered bacteria comprising a uric acid degradation enzyme further comprise a kill-switch circuit, such as any of the kill-switch circuits provided herein.
  • the genetically engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter, and an inverted toxin sequence.
  • the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.
  • the engineered bacteria further comprise one or more genes encoding one or more recombinase(s) under the control of an inducible promoter and one or more inverted excision genes, wherein the excision gene(s) encode an enzyme that deletes an essential gene.
  • the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.
  • the engineered bacteria further comprise one or more genes encoding a toxin under the control of a promoter having a TetR repressor binding site and a gene encoding the TetR under the control of an inducible promoter that is induced by arabinose, such as ParaBAD.
  • the genetically engineered bacteria further comprise one or more genes encoding an antitoxin.
  • the genetically engineered bacterium is an auxotroph comprising a uric acid degradation enzyme gene and further comprises a kill-switch circuit, such as any of the kill- switch circuits described herein.
  • the gene encoding a uric acid degradation enzyme is present on a plasmid in the bacterium and operatively linked on the plasmid to the promoter that is induced under low-oxygen or anaerobic conditions.
  • the gene encoding a uric acid degradation enzyme is present in the bacterial chromosome and is operatively linked in the chromosome to the promoter that is induced under low- oxygen or anaerobic conditions.
  • A. Uric Acid Catabolism Enzymes [0183] As used herein, the term “uric acid catabolism enzyme” or “uric acid degradation enzyme” refers to an enzyme involved in the catabolism, processing, degradation, or breakdown of uric acid. Enzymes involved in the catabolism of uric acid may be expressed or modified in the cells disclosed herein in order to enhance degradation of uric acid.
  • the cells when at least one uric acid degradation enzyme is expressed in the host cells, e.g., bacterial cells, disclosed herein, the cells convert more of the target uric acid into one or more byproducts when the enzyme is expressed than unmodified cells of the same bacterial subtype under the same conditions.
  • the host cells such as a bacteria, comprising a heterologous gene encoding at least one uric acid degradation enzyme can degrade the target uric acid to treat a disease and/or disorder.
  • the uric acid catabolism enzyme is a uricase.
  • the uric acid catabolism enzyme degrades uric acid.
  • the uric acid catabolism enzyme increases the rate of degradation of uric acid in the cell. In one embodiment, the uric acid catabolism enzyme decreases the level of uric acid in the cell or in the subject. In another embodiment, the uric acid catabolism enzyme increases the level of uric acid byproduct in the cell or in the subject as compared to the level of the uric acid in the cell or in the subject.
  • the recombinant host cell such as a host bacterial cell, comprises a heterologous gene encoding a uric acid catabolism enzyme.
  • the disclosure provides a host cell, such as a host bacterial cell, that comprises a heterologous gene encoding a uric acid catabolism enzyme operably linked to a first promoter, e.g., an inducible promoter or a constitutive promoter.
  • the host cell such as a host bacterial cell, comprises gene encoding a uric acid catabolism enzyme from a different organism, e.g., a different species of bacteria.
  • the host cell such as a host bacterial cell, comprises more than one copy of a native gene encoding a uric acid catabolism enzyme.
  • the host cell such as a host bacterial cell, comprises a native gene encoding a uric acid catabolism enzyme, as well as at least one copy of a gene encoding a uric acid catabolism enzyme from a different organism, e.g., a different species of bacteria.
  • the host cell such as a host bacterial cell, comprises at least one, two, three, four, five, or six copies of a gene encoding a uric acid catabolism enzyme.
  • the host cell such as a host bacterial cell, comprises multiple copies of a gene encoding a uric acid catabolism enzyme.
  • the host cell e.g., bacteria, yeast, plant, animal, or protozoa cell
  • the host cell comprises a heterologous gene encoding a uric acid catabolism enzyme, wherein said uric acid catabolism enzyme comprises an amino acid sequence that has at least 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of a polypeptide encoded by a uric acid catabolism enzyme gene disclosed herein.
  • Multiple distinct uric acid catabolism enzymes are known in the art.
  • uric acid catabolism enzyme is encoded by a gene encoding a uric acid catabolism enzyme derived from a bacterial species. In some embodiments, a uric acid catabolism enzyme is encoded by a gene encoding a uric acid catabolism enzyme derived from a non-bacterial species. In some embodiments, a uric acid catabolism enzyme is encoded by a gene derived from a eukaryotic species, e.g., protozoan species, a fungal species, a yeast species, or a plant species. In one embodiment, a uric acid catabolism enzyme is encoded by a gene derived from a human.
  • the gene encoding the uric acid catabolism enzyme is derived from an organism of the genus or species that includes, but is not limited to, Acetinobacter, Azospirillum, Bacillus, Bacteroides, Bifidobacterium, Brevibacteria, Burkholderia, Citrobacter, Clostridium, Corynebacterium, Cronobacter, Enterobacter, Enterococcus, Erwinia, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Leishmania, Listeria, Macrococcus, Mycobacterium, Nakamurella, Nasonia, Nostoc, Pantoea, Pectobacterium, Pseudomonas, Psychrobacter, Ralstonia, Saccharomyces, Salmonella, Sarcina, Serratia, Staphylococcus, and Yersinia, e.g., Acetinobacter radioresistens, Acetinobacter, Azo
  • the at least one gene encoding the at least one uric acid catabolism enzyme is derived from an organism of the genus or species that includes, but is not limited to, Achromobacter parvulus, Acidomonas methanolica, Agrobacterium tumefaciens, Aminobacter aminovorans, Ancylobacter aquaticus, Arthrobacter spp., Bacillus spp., such as Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus methanolicus, Bacillus halodurans, or Bacillus subtilis, Beggiatoa alba, Ceriporiopsis subvermispora, Clostridium botulinum, Clostridium carboxidivorans, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus oxalaticus, Desulfovibrio desulfuricans, Escherichia coli, Flavobacterium
  • the at least one gene encoding the at least one uric acid catabolism enzyme is derived from an organism of the genus or species that includes, but is not limited to Arabidopsis thaliana, Candida spp., such as Candida boidinii, Candida methanolica, or Candida methylica, Saccharomyces cerevisiae, or Torulopsis candida.
  • the at least one gene encoding the at least one uric acid catabolism enzyme is derived from an organism of the genus or species that includes, but is not limited to, Bifidobacterium, Bordetella, Bradyrhizobium, Burkholderia, Clostridium, Enterococcus, Escherichia, Eubacterium, Lactobacillus, Magnetospirillium, Mycobacterium, Neurospora, Oxalobacter, Ralstonia, Rhodopseudomonas, Shigella, Thermoplasma, and Thauera, e.g., Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bordatella bronchiseptica, Bordatella parapertussis, Burkholderia fungorum, Burkholderia xenovor
  • the uricase gene has at least about 80% identity with the sequence of any one of SEQ ID NOs: 3, 209, 210, 225-227, 260, 262, 264, 266, 268, 270, or 272. Accordingly, in one embodiment, the uricase gene has at least about 90% identity with the sequence of any one of SEQ ID NOs: 3, 209, 210, 225-227, 260, 262, 264, 266, 268, 270, or 272. Accordingly, in one embodiment, the uricase gene has at least about 95% identity with the sequence of any one of SEQ ID NOs: 3, 209, 210, 225-227, 260, 262, 264, 266, 268, 270, or 272.
  • the uricase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of any one of SEQ ID NOs: 3, 209, 210, 225-227, 260, 262, 264, 266, 268, 270, or 272.
  • the uricase gene comprises the sequence of any one of SEQ ID NOs: 3, 209, 210, 225-227, 260, 262, 264, 266, 268, 270, or 272.
  • the uricase gene consists of the sequence of any one of SEQ ID NOs: 3, 209, 210, 225-227, 260, 262, 264, 266, 268, 270, or 272.
  • the uricase polypeptide has at least about 80% identity with the sequence of any one of SEQ ID NOs: 8, 218, 219, 228,-230, 261, 263, 265, 267, 269, 271, or 273.
  • the uricase polypeptide has at least about 90% identity with the sequence of any one of SEQ ID NOs: 8, 218, 219, 228,-230, 261, 263, 265, 267, 269, 271, or 273.
  • the uricase polypeptide has at least about 95% identity with the sequence of any one of SEQ ID NOs: 8, 218, 219, 228,-230, 261, 263, 265, 267, 269, 271, or 273. Accordingly, in one embodiment, the uricase polypeptide has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of any one of SEQ ID NOs: 8, 218, 219, 228,-230, 261, 263, 265, 267, 269, 271, or 273.
  • the uricase polypeptide comprises the sequence of any one of SEQ ID NOs: 8, 218, 219, 228,-230, 261, 263, 265, 267, 269, 271, or 273.
  • the uricase polypeptide consists of the sequence of any one of SEQ ID NOs: 8, 218, 219, 228,-230, 261, 263, 265, 267, 269, 271, or 273.
  • the uric acid catabolism enzyme is urate oxidase (uricase).
  • the uricase gene is a gene from Candida utilis.
  • the uricase gene has at least about 80% identity with the sequence of SEQ ID NO: 3 or SEQ ID NO: 225.
  • the uricase gene has at least about 90% identity with the sequence of SEQ ID NO: 3 or SEQ ID NO: 225. Accordingly, in one embodiment, the uricase gene has at least about 95% identity with the sequence of SEQ ID NO: 3 or SEQ ID NO: 225. Accordingly, in one embodiment, the uricase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 3 or SEQ ID NO: 225. In another embodiment, the uricase gene comprises the sequence of SEQ ID NO: 3 or SEQ ID NO: 225.
  • the uricase gene consists of the sequence of SEQ ID NO: 3 or SEQ ID NO: 225.
  • the uricase gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 8 or SEQ ID NO: 228. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 8 or SEQ ID NO: 228. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 8 or SEQ ID NO: 228.
  • the uricase gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 8 or SEQ ID NO: 228.
  • the uricase gene encodes a protein comprising the sequence of SEQ ID NO: 8 or SEQ ID NO: 228.
  • the uricase gene encodes a protein consisting of the sequence of SEQ ID NO: 8 or SEQ ID NO: 228.
  • the uric acid catabolism enzyme is urate oxidase (uricase).
  • the uricase gene is a gene from Candida utilis. In one embodiment, the uricase gene has at least about 80% identity with the sequence of SEQ ID NO: 226. Accordingly, in one embodiment, the uricase gene has at least about 90% identity with the sequence of SEQ ID NO: 226. Accordingly, in one embodiment, the uricase gene has at least about 95% identity with the sequence of SEQ ID NO: 226. Accordingly, in one embodiment, the uricase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 226.
  • the uricase gene comprises the sequence of SEQ ID NO: 226. In yet another embodiment, the uricase gene consists of the sequence of SEQ ID NO: 226. [0195] In one embodiment, the uricase gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 229. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 229. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 229.
  • the uricase gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 229.
  • the uricase gene encodes a protein comprising the sequence of SEQ ID NO: 229.
  • the uricase gene encodes a protein consisting of the sequence of SEQ ID NO: 229.
  • the uric acid catabolism enzyme is urate oxidase (uricase).
  • the uricase gene is a gene from Mus musculus.
  • the uricase gene has at least about 80% identity with the sequence of SEQ ID NO: 227. Accordingly, in one embodiment, the uricase gene has at least about 90% identity with the sequence of SEQ ID NO: 227. Accordingly, in one embodiment, the uricase gene has at least about 95% identity with the sequence of SEQ ID NO: 227. Accordingly, in one embodiment, the uricase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 227. In another embodiment, the uricase gene comprises the sequence of SEQ ID NO: 227.
  • the uricase gene consists of the sequence of SEQ ID NO: 227. [0197] In one embodiment, the uricase gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 230. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 230. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 230.
  • the uricase gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 230.
  • the uricase gene encodes a protein comprising the sequence of SEQ ID NO: 230.
  • the uricase gene encodes a protein consisting of the sequence of SEQ ID NO: 230.
  • the uric acid catabolism enzyme is urate oxidase (uricase).
  • the uricase gene is a gene from Arthrobacter globiformis.
  • the uricase gene has at least about 80% identity with the sequence of SEQ ID NO: 209. Accordingly, in one embodiment, the uricase gene has at least about 90% identity with the sequence of SEQ ID NO: 209. Accordingly, in one embodiment, the uricase gene has at least about 95% identity with the sequence of SEQ ID NO: 209. Accordingly, in one embodiment, the uricase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 209. In another embodiment, the uricase gene comprises the sequence of SEQ ID NO: 209.
  • the uricase gene consists of the sequence of SEQ ID NO: 209. [0199] In one embodiment, the uricase gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 218. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 218. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 218.
  • the uricase gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 218.
  • the uricase gene encodes a protein comprising the sequence of SEQ ID NO: 218.
  • the uricase gene encodes a protein consisting of the sequence of SEQ ID NO: 218.
  • the uric acid catabolism enzyme is urate oxidase (uricase).
  • the uricase gene is a gene from Chimaeribacter californicus.
  • the uricase gene has at least about 80% identity with the sequence of SEQ ID NO: 210. Accordingly, in one embodiment, the uricase gene has at least about 90% identity with the sequence of SEQ ID NO: 210. Accordingly, in one embodiment, the uricase gene has at least about 95% identity with the sequence of SEQ ID NO: 210. Accordingly, in one embodiment, the uricase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 210. In another embodiment, the uricase gene comprises the sequence of SEQ ID NO: 210.
  • the uricase gene consists of the sequence of SEQ ID NO: 210. [0201] In one embodiment, the uricase gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 219. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 219. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 219.
  • the uricase gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 219.
  • the uricase gene encodes a protein comprising the sequence of SEQ ID NO: 219.
  • the uricase gene encodes a protein consisting of the sequence of SEQ ID NO: 219.
  • the uric acid catabolism enzyme is urate oxidase (uricase).
  • the uricase gene is a gene from Cyberlindnera jadinii.
  • the uricase gene has at least about 80% identity with the sequence of SEQ ID NO: 260. Accordingly, in one embodiment, the uricase gene has at least about 90% identity with the sequence of SEQ ID NO: 260. Accordingly, in one embodiment, the uricase gene has at least about 95% identity with the sequence of SEQ ID NO: 260. Accordingly, in one embodiment, the uricase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 260. In another embodiment, the uricase gene comprises the sequence of SEQ ID NO: 260.
  • the uricase gene consists of the sequence of SEQ ID NO: 260. [0203] In one embodiment, the uricase gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 261. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 261. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 261.
  • the uricase gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 261.
  • the uricase gene encodes a protein comprising the sequence of SEQ ID NO: 261.
  • the uricase gene encodes a protein consisting of the sequence of SEQ ID NO: 261.
  • the uric acid catabolism enzyme is urate oxidase (uricase).
  • the uricase gene is a gene from Methylobacterium oxalidis.
  • the uricase gene has at least about 80% identity with the sequence of SEQ ID NO: 262. Accordingly, in one embodiment, the uricase gene has at least about 90% identity with the sequence of SEQ ID NO: 262. Accordingly, in one embodiment, the uricase gene has at least about 95% identity with the sequence of SEQ ID NO: 262. Accordingly, in one embodiment, the uricase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 262. In another embodiment, the uricase gene comprises the sequence of SEQ ID NO: 262.
  • the uricase gene consists of the sequence of SEQ ID NO: 262. [0205] In one embodiment, the uricase gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 263. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 263. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 263.
  • the uricase gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 263.
  • the uricase gene encodes a protein comprising the sequence of SEQ ID NO: 263.
  • the uricase gene encodes a protein consisting of the sequence of SEQ ID NO: 263.
  • the uric acid catabolism enzyme is urate oxidase (uricase).
  • the uricase gene is a gene from Bosea sp. RCAM04685.
  • the uricase gene has at least about 80% identity with the sequence of SEQ ID NO: 264. Accordingly, in one embodiment, the uricase gene has at least about 90% identity with the sequence of SEQ ID NO: 264. Accordingly, in one embodiment, the uricase gene has at least about 95% identity with the sequence of SEQ ID NO: 264. Accordingly, in one embodiment, the uricase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 264. In another embodiment, the uricase gene comprises the sequence of SEQ ID NO: 264.
  • the uricase gene consists of the sequence of SEQ ID NO: 264. [0207] In one embodiment, the uricase gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 265. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 265. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 265.
  • the uricase gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 265.
  • the uricase gene encodes a protein comprising the sequence of SEQ ID NO: 265.
  • the uricase gene encodes a protein consisting of the sequence of SEQ ID NO: 265.
  • the uric acid catabolism enzyme is urate oxidase (uricase).
  • the uricase gene is a gene from Cryptococcus neoformans var.
  • the uricase gene has at least about 80% identity with the sequence of SEQ ID NO: 266. Accordingly, in one embodiment, the uricase gene has at least about 90% identity with the sequence of SEQ ID NO: 266. Accordingly, in one embodiment, the uricase gene has at least about 95% identity with the sequence of SEQ ID NO: 266. Accordingly, in one embodiment, the uricase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 266. In another embodiment, the uricase gene comprises the sequence of SEQ ID NO: 266.
  • the uricase gene consists of the sequence of SEQ ID NO: 266. [0209] In one embodiment, the uricase gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 267. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 267. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 267.
  • the uricase gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 267.
  • the uricase gene encodes a protein comprising the sequence of SEQ ID NO: 267.
  • the uricase gene encodes a protein consisting of the sequence of SEQ ID NO: 267.
  • the uric acid catabolism enzyme is urate oxidase (uricase).
  • the uricase gene is a gene from Methylobacterium sp. Leaf104.
  • the uricase gene has at least about 80% identity with the sequence of SEQ ID NO: 268. Accordingly, in one embodiment, the uricase gene has at least about 90% identity with the sequence of SEQ ID NO: 268. Accordingly, in one embodiment, the uricase gene has at least about 95% identity with the sequence of SEQ ID NO: 268. Accordingly, in one embodiment, the uricase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 268. In another embodiment, the uricase gene comprises the sequence of SEQ ID NO: 268.
  • the uricase gene consists of the sequence of SEQ ID NO: 268. [0211] In one embodiment, the uricase gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 269. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 269. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 269.
  • the uricase gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 269.
  • the uricase gene encodes a protein comprising the sequence of SEQ ID NO: 269.
  • the uricase gene encodes a protein consisting of the sequence of SEQ ID NO: 269.
  • the uric acid catabolism enzyme is urate oxidase (uricase).
  • the uricase gene is a gene from Alphaproteobacteria bacterium.
  • the uricase gene has at least about 80% identity with the sequence of SEQ ID NO: 270. Accordingly, in one embodiment, the uricase gene has at least about 90% identity with the sequence of SEQ ID NO: 270. Accordingly, in one embodiment, the uricase gene has at least about 95% identity with the sequence of SEQ ID NO: 270. Accordingly, in one embodiment, the uricase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 270. In another embodiment, the uricase gene comprises the sequence of SEQ ID NO: 270.
  • the uricase gene consists of the sequence of SEQ ID NO: 270. [0213] In one embodiment, the uricase gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 271. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 271. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 271.
  • the uricase gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 271.
  • the uricase gene encodes a protein comprising the sequence of SEQ ID NO: 271.
  • the uricase gene encodes a protein consisting of the sequence of SEQ ID NO: 271.
  • the uric acid catabolism enzyme is urate oxidase (uricase).
  • the uricase gene is a gene from Paenarthrobacter nicotinovorans.
  • the uricase gene has at least about 80% identity with the sequence of SEQ ID NO: 272. Accordingly, in one embodiment, the uricase gene has at least about 90% identity with the sequence of SEQ ID NO: 272. Accordingly, in one embodiment, the uricase gene has at least about 95% identity with the sequence of SEQ ID NO: 272. Accordingly, in one embodiment, the uricase gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 272. In another embodiment, the uricase gene comprises the sequence of SEQ ID NO: 272.
  • the uricase gene consists of the sequence of SEQ ID NO: 272. [0215] In one embodiment, the uricase gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 273. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 273. Accordingly, in one embodiment, the uricase gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 273.
  • the uricase gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 273.
  • the uricase gene encodes a protein comprising the sequence of SEQ ID NO: 273.
  • the uricase gene encodes a protein consisting of the sequence of SEQ ID NO: 273.
  • the uricase gene encodes a polypeptide that has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 point mutations as compared to wildtype uricase polypeptide.
  • the uricase gene encodes a polypeptides that has one or more point mutations selected from F275M and A346S as compared to a wild type polypeptide.
  • the sequence of a uricase associated with the disclosure comprises one or more amino acid substitutions relative to SEQ ID NO: 10 or 221.
  • the one or more amino acid substations are at a position corresponding to position 275 and/or 346 in SEQ ID NO: 10 or 221.
  • the uricase comprises a methionine (M) at a position corresponding to position 275 in SEQ ID NO: 10 or 221; a serine (S) at a position corresponding to position 346 in SEQ ID NO: 10 or 221.
  • the sequence of a uricase associated with the disclosure comprises substitutions at a position or responding to: position 275 and/or position 346 in the sequence of SEQ ID NO: 10 or 221.
  • the sequence of a uricase comprises the following amino acid substitutions relative to the sequences of SEQ ID NO: 10 or 221: F275M and/or A346S.
  • the uricase is an Aspergillus flavus rasburicase. In one embodiment, the uricase is EC 1.7.3.3. In one embodiment, the uricase is a Candida utilis uricase. In one embodiment, the uricase is an E. coli uricase. In one embodiment, the uricase is a C. californicus uricase. In one embodiment, the uricase is an A. globiformis uricase. [0221] In one embodiment, the uric acid catabolism enzyme is uncharacterized protein ygfT (ygfT). In one embodiment, the ygfT gene is a gene from E. coli.
  • the ygfT gene has at least about 80% identity with the sequence of SEQ ID NO: 4. Accordingly, in one embodiment, the ygfT gene has at least about 90% identity with the sequence of SEQ ID NO: 4. Accordingly, in one embodiment, the ygfT gene has at least about 95% identity with the sequence of SEQ ID NO: 4. Accordingly, in one embodiment, the ygfT gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 4. In another embodiment, the ygfT gene comprises the sequence of SEQ ID NO: 4.
  • the ygfT gene consists of the sequence of SEQ ID NO: 4. [0222] In one embodiment, the ygfT gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 199. Accordingly, in one embodiment, the ygfT gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 199. Accordingly, in one embodiment, the ygfT gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 199.
  • the ygfT gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 199.
  • the ygfT gene encodes a protein comprising the sequence of SEQ ID NO: 199.
  • the ygfT gene encodes a protein consisting of the sequence of SEQ ID NO: 199. [0223]
  • the ygfT is EC 1.18.1.2.
  • the at least one gene encoding the at least one uric acid catabolism enzyme has been codon-optimized for use in the recombinant bacterial cell disclosed herein. In one embodiment, the at least one gene encoding the at least one uric acid catabolism enzyme has been codon-optimized for use in Escherichia coli. In another embodiment, the at least one gene encoding the at least one uric acid catabolism enzyme has been codon-optimized for use in Lactococcus.
  • the at least one gene encoding the at least one uric acid catabolism enzyme is expressed in the recombinant bacterial cells disclosed herein, the bacterial cells catabolize more of the target uric acid than unmodified bacteria of the same bacterial subtype under the same conditions (e.g., culture or environmental conditions).
  • the genetically engineered bacteria comprising at least one heterologous gene encoding at least one uric acid catabolism enzyme may be used to catabolize uric acid in order to treat a disease and/or disorder associated with uric acid, e.g., hyperuricemia and/or gout.
  • the present disclosure further provides genes encoding functional fragments of at least one uric acid catabolism enzyme or functional variants of at least one uric acid catabolism enzyme.
  • the term “functional fragment thereof” or “functional variant thereof” of at least one uric acid catabolism enzyme relates to an element having qualitative biological activity in common with the wild-type uric acid catabolism enzyme from which the fragment or variant was derived (e.g., a domain of the uric acid catabolism enzyme).
  • a functional fragment or a functional variant of a mutated uric acid catabolism enzyme is one which retains essentially the same ability to catabolize uric acid as the uric acid catabolism enzyme from which the functional fragment or functional variant was derived.
  • a polypeptide having uric acid catabolism enzyme activity may be truncated at the N-terminus or C-terminus and the retention of uric acid catabolism enzyme activity assessed using assays known to those of skill in the art, including the exemplary assays provided herein.
  • the recombinant bacterial cell disclosed herein comprises a heterologous gene encoding at least one uric acid catabolism enzyme functional variant.
  • the recombinant bacterial cell disclosed herein comprises a heterologous gene encoding at least one uric acid catabolism enzyme functional fragment.
  • the gene encoding a uric acid catabolism enzyme is mutagenized; mutants exhibiting increased activity are selected; and the mutagenized gene encoding the uric acid catabolism enzyme is isolated and inserted into the bacterial cell described herein.
  • spontaneous mutants that arise that allow bacteria to grow on amino acids as the sole carbon source can be screened for and selected.
  • the gene comprising the modifications described herein may be present on a plasmid or chromosome.
  • percent (%) sequence identity or “percent (%) identity,” also including “homology,” is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • Optimal alignment of the sequences for comparison may be produced, besides manually, by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math.2, 482, by means of the local homology algorithm of Needleman and Wunsch, 1970, J. Mol.
  • the present disclosure encompasses genes encoding at least one uric acid catabolism enzyme comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein.
  • Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions.
  • a conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g., charge, structure, polarity, hydrophobicity/hydrophilicity) that are similar to those of the first amino acid.
  • Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; C, S and T.
  • a basic amino acid with another basic amino acid (e.g., replacement among Lys, Arg, His), replacing an acidic amino acid with another acidic amino acid (e.g., replacement among Asp and Glu), replacing a neutral amino acid with another neutral amino acid (e.g., replacement among Ala, Gly, Ser, Met, Thr, Leu, Ile, Asn, Gln, Phe, Cys, Pro, Trp, Tyr, Val).
  • Assays for testing the activity of a uric acid catabolism enzyme, a uric acid catabolism enzyme functional variant, or a uric acid catabolism enzyme functional fragment are well known to one of ordinary skill in the art.
  • uric acid catabolism can be assessed by expressing the protein, functional variant, or fragment thereof, in a recombinant bacterial cell that lacks endogenous uric acid catabolism enzyme activity.
  • Uric acid catabolism can be assessed using the assay method as described by Ihler et al., J. Clin. Invest, 56(3):595-602, 2008).
  • catabolism of uric acid can also be assessed in vitro by measuring the disappearance of uric acid as described by Lee (see, for example, Lee et al., BMC Complement Altern Med, 19(1):57, 2019). Additional assays are described in detail in the uric acid catabolism enzyme subsections, below.
  • the host cell such as a bacterial cell, disclosed herein comprises at least one heterologous gene encoding at least one uric acid catabolism enzyme.
  • the recombinant host cells, such as host bacterial cells, described herein comprise one uric acid catabolism enzyme.
  • the recombinant host cells, such as host bacterial cells, described herein comprise two uric acid catabolism enzymes.
  • the recombinant host cells, such as host bacterial cells, described herein comprise three uric acid catabolism enzymes.
  • the recombinant host cells, such as host bacterial cells, described herein comprise four uric acid catabolism enzymes.
  • the recombinant host cells such as host bacterial cells, described herein comprise five uric acid catabolism enzymes.
  • the disclosure provides a host cell, such as a bacterial cell, that comprises at least one heterologous gene encoding at least one uric acid catabolism enzyme operably linked to a first promoter.
  • the first promoter is an inducible promoter.
  • the first promoter is a constitutive promoter.
  • the host cell, such as a bacterial cell comprises at least one gene encoding at least one uric acid catabolism enzyme from a different organism, e.g., a different species of bacteria.
  • the host cell such as a bacterial cell, comprises more than one copy of a native gene encoding at least one uric acid catabolism enzyme.
  • the host cell such as a bacterial cell, comprises at least one native gene encoding at least one uric acid catabolism enzyme, as well as at least one copy of at least one gene encoding at least one uric acid catabolism enzyme from a different organism, e.g., a different species of bacteria.
  • the host cell such as a bacterial cell, comprises at least one, two, three, four, five, or six copies of a gene encoding at least one uric acid catabolism enzyme.
  • the host cell such as a bacterial cell, comprises multiple copies of a gene or genes encoding at least one uric acid catabolism enzyme.
  • the gene encoding the uric acid catabolism enzyme is directly operably linked to a first promoter.
  • the gene encoding the uric acid catabolism enzyme is indirectly operably linked to a first promoter.
  • the gene encoding the uric acid catabolism enzyme is operably linked to a promoter that is not associated with the uric acid catabolism gene in nature.
  • the gene encoding the uric acid catabolism enzyme is expressed under the control of a constitutive promoter.
  • the gene encoding the uric acid catabolism enzyme is expressed under the control of an inducible promoter. In some embodiments, the gene encoding the uric acid catabolism enzyme is expressed under the control of a promoter that is directly or indirectly induced by exogenous environmental conditions. In one embodiment, the gene encoding the uric acid catabolism enzyme is expressed under the control of a promoter that is directly or indirectly induced by low-oxygen or anaerobic conditions, wherein expression of the gene encoding the uric acid catabolism enzyme is activated under low-oxygen or anaerobic environments, such as the environment of the mammalian gut. Inducible promoters are described in more detail infra.
  • the gene encoding the uric acid catabolism enzyme may be present on a plasmid or chromosome in the bacterial cell. In one embodiment, the gene encoding the uric acid catabolism enzyme is located on a plasmid in the bacterial cell. In another embodiment, the gene encoding the uric acid catabolism enzyme is located in the chromosome of the bacterial cell. In yet another embodiment, a native copy of the gene encoding the uric acid catabolism enzyme is located in the chromosome of the bacterial cell, and a gene encoding a uric acid catabolism enzyme from a different species of bacteria is located on a plasmid in the bacterial cell.
  • a native copy of the gene encoding the uric acid catabolism enzyme is located on a plasmid in the bacterial cell, and a gene encoding the uric acid catabolism enzyme from a different species of bacteria is located on a plasmid in the bacterial cell.
  • a native copy of the gene encoding the uric acid catabolism enzyme is located in the chromosome of the bacterial cell, and a gene encoding the uric acid catabolism enzyme from a different species of bacteria is located in the chromosome of the bacterial cell.
  • the gene encoding the uric acid catabolism enzyme is expressed on a low-copy plasmid.
  • the gene encoding the uric acid catabolism enzyme is expressed on a high-copy plasmid.
  • the high-copy plasmid may be useful for increasing expression of the uric acid catabolism enzyme, thereby increasing the catabolism of the uric acid.
  • a recombinant bacterial cell comprising the gene encoding the uric acid catabolism enzyme expressed on a high-copy plasmid does not increase uric acid catabolism or decrease uric acid levels as compared to a recombinant bacterial cell comprising the same gene expressed on a low-copy plasmid in the absence of a heterologous transporter of the uric acid and additional copies of a native transporter of the uric acid. It has been surprisingly discovered that in some embodiments, the rate-limiting step of uric acid catabolism is not expression of a uric acid catabolism enzyme, but rather availability of the uric acid.
  • uric acid transport into the cell it may be advantageous to increase uric acid transport into the cell, thereby enhancing uric acid catabolism.
  • the inventors of the instant application have surprisingly found that, in conjunction with overexpression of a transporter of uric acid even low copy number plasmids comprising a gene encoding a uric acid catabolism enzyme are capable of almost completely eliminating uric acid from a sample.
  • a transporter of uric acid into the recombinant bacterial cell
  • a low-copy plasmid comprising the gene encoding the uric acid catabolism enzyme in conjunction in order to enhance the stability of expression of the uric acid catabolism enzyme, while maintaining high uric acid catabolism and to reduce negative selection pressure on the transformed bacterium.
  • the uric acid transporter is used in conjunction with a high-copy plasmid.
  • a recombinant bacterium capable of degrading uric acid at a rate of 0.3 umol/hour/1e9 cells. In one embodiment, disclosed herein is a recombinant bacterium capable of degrading uric acid at a rate of 0.74 umol/hour/1e9 cells.
  • a recombinant bacterium capable of degrading uric acid at a rate of at least 0.1 umol/hour/1e9 cells, at least 0.2 umol/hour/1e9 cells, at least 0.3 umol/hour/1e9 cells, at least 0.4 umol/hour/1e9 cells, at least 0.5 umol/hour/1e9 cells, at least 0.6 umol/hour/1e9 cells, at least 0.7 umol/hour/1e9 cells, at least 0.8 umol/hour/1e9 cells, or at least 0.9 umol/hour/1e9 cells.
  • a recombinant bacterium capable of degrading uric acid at a rate of about 0.1 umol/hour/1e9 cells to about 0.9 umol/hour/1e9 cells, about 0.2 umol/hour/1e9 cells to about 0.9 umol/hour/1e9 cells, about 0.3 umol/hour/1e9 cells to about 0.9 umol/hour/1e9 cells, about 0.4 umol/hour/1e9 cells to about 0.9 umol/hour/1e9 cells, about 0.5 umol/hour/1e9 cells to about 0.9 umol/hour/1e9 cells, about 0.6 umol/hour/1e9 cells to about 0.9 umol/hour/1e9 cells, about 0.7 umol/hour/1e9 cells to about 0.9 umol/hour/1e9 cells, about 0.8 umol/hour/1e9 cells to about 0.9 umol/hour/1e9 cells, about 0.1 umol/hour/1e9
  • the uricase enzyme is active in the presence of oxygen. In another embodiment, the uricase enzyme is active in the absence of oxygen.
  • Multiple distinct uric acid degrading enzymes are well known in the art and are described, below.
  • B. Transporters [0242] The uptake of urate into bacterial cells is mediated by proteins well known to those of skill in the art. Urate transporters, i.e., importers, (also referred to herein as “uric acid transporters” or “uric acid importers”) may be expressed or modified in the bacteria in order to enhance urate transport into the cell.
  • the bacterial cells import more urate into the cell when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the genetically engineered host cell such as a bacterial cell, comprising a heterologous gene encoding a transporter of urate, which may be used to import urate into the host cell, such as a bacterial cell, so that any gene encoding a uric acid catabolism enzyme expressed in the organism, e.g., co-expressed uric acid catabolism enzyme, can catabolize the uric acid to treat diseases associated with uric acid, such as hyperuricemia.
  • the host cell such as a bacterial cell, comprises a heterologous gene encoding one or more transporter(s) of urate.
  • the host cell such as a bacterial cell, comprises a heterologous gene encoding a transporter of uric acid and a heterologous gene encoding one or more uric acid catabolism enzymes.
  • the host cell such as a bacterial cell, comprises a heterologous gene encoding an importer of uric acid and a genetic modification that reduces export of uric acid, e.g., a genetic mutation in an exporter gene or promoter.
  • the host cell such as a bacterial cell, comprises a heterologous gene encoding an importer of uric acid, a heterologous gene encoding a uric acid catabolism enzyme, and a genetic modification that reduces export of uric acid.
  • a host cell e.g., bacterial cell, that comprises a heterologous gene encoding a uric acid catabolism enzyme operably linked to a first promoter and at least one heterologous gene encoding a transporter of uric acid.
  • a host cell such as a bacterial cell, that comprises at least one heterologous gene encoding a transporter of uric acid operably linked to the first promoter.
  • a host cell such as a bacterial cell, that comprises a heterologous gene encoding a uric acid catabolism enzyme operably linked to a first promoter and at least one heterologous gene encoding a transporter of uric acid operably linked to a second promoter.
  • the first promoter and the second promoter are separate copies of the same promoter.
  • the first promoter and the second promoter are different promoters.
  • the bacterial cell comprises at least one gene encoding a transporter of uric acid from a different organism, e.g., a different species of bacteria.
  • the host cell such as a bacterial cell, comprises at least one native gene encoding a transporter of uric acid.
  • the at least one native gene encoding a transporter of uric acid is not modified.
  • the host cell such as a bacterial cell, comprises more than one copy of at least one native gene encoding a transporter of uric acid.
  • the host cell such as a bacterial cell, comprises a copy of at least one gene encoding a native transporter of uric acid, as well as at least one copy of at least one heterologous gene encoding a transporter of uric acid from a different bacterial species.
  • the host cell such as a bacterial cell, comprises at least one, two, three, four, five, or six copies of the at least one heterologous gene encoding a transporter of uric acid.
  • the host cell such as a bacterial cell, comprises multiple copies of the at least one heterologous gene encoding a transporter of uric acid.
  • the recombinant host cell such as a bacterial cell, comprises a heterologous gene encoding a uric acid transporter, wherein said transporter comprises an amino acid sequence that has at least 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of a polypeptide encoded by a uric acid transporter gene disclosed herein.
  • the transporter is encoded by a transporter of a uric acid gene derived from a bacterial genus or species, including but not limited to, Bacillus, Campylobacter, Clostridium, Escherichia, Lactobacillus, Pseudomonas, Salmonella, Staphylococcus, Bacillus subtilis, Campylobacter jejuni, Clostridium perfringens, Escherichia coli, Lactobacillus delbrueckii, Pseudomonas aeruginosa, Salmonella typhimurium, or Staphylococcus aureus.
  • the bacterial species is Escherichia coli.
  • the bacterial species is Escherichia coli strain Nissle.
  • the present disclosure further comprises genes encoding functional fragments of a transporter of uric acid or functional variants of a transporter of uric acid.
  • functional fragment thereof or “functional variant thereof” of a transporter of uric acid relates to an element having qualitative biological activity in common with the wild-type transporter of uric acid from which the fragment or variant was derived.
  • a functional fragment or a functional variant of a mutated transporter of uric acid is one which retains essentially the same ability to import uric acid into the host cell, such as a bacterial cell, as does the transporter protein from which the functional fragment or functional variant was derived.
  • the recombinant host cell such as a bacterial cell, comprises at least one heterologous gene encoding a functional fragment of a transporter of uric acid.
  • the recombinant host cell such as a bacterial cell, comprises a heterologous gene encoding a functional variant of a transporter of uric acid.
  • Assays for testing the activity of a transporter of uric acid, a functional variant of a transporter of uric acid, or a functional fragment of transporter of uric acid are well known to one of ordinary skill in the art.
  • import of uric acid may be determined using the methods as described in Haney et al., J. Bact., 174(1):108-15, 1992; Rahmanian et al., J. Bact., 116(3):1258-66, 1973; and Ribardo and Hendrixson, J. Bact., 173(22):6233-43, 2011, the entire contents of each of which are expressly incorporated by reference herein.
  • the genes encoding the transporter of uric acid have been codon- optimized for use in the host organism. In one embodiment, the genes encoding the transporter of uric acid have been codon-optimized for use in Escherichia coli. [0250] The present disclosure also encompasses genes encoding a transporter of uric acid comprising amino acids in its sequence that are substantially the same as an amino acid sequence described herein. Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions.
  • the at least one gene encoding a transporter of uric acid is mutagenized; mutants exhibiting increased uric acid transport are selected; and the mutagenized at least one gene encoding a transporter of uric acid is isolated and inserted into the bacterial cell.
  • the at least one gene encoding a transporter of uric acid is mutagenized; mutants exhibiting decreased uric acid transport are selected; and the mutagenized at least one gene encoding a transporter of uric acid is isolated and inserted into the bacterial cell.
  • the transporter modifications described herein may be present on a plasmid or chromosome.
  • the host cell such as a bacterial cell, comprises a heterologous gene encoding a uric acid catabolism enzyme operably linked to a first promoter and at least one heterologous gene encoding a transporter of uric acid.
  • the at least one heterologous gene encoding a transporter of uric acid is operably linked to the first promoter.
  • the at least one heterologous gene encoding a transporter of uric acid is operably linked to a second promoter.
  • the at least one gene encoding a transporter of uric acid is directly operably linked to the second promoter.
  • the at least one gene encoding a transporter of uric acid is indirectly operably linked to the second promoter.
  • expression of at least one gene encoding a transporter of uric acid is controlled by a different promoter than the promoter that controls expression of the gene encoding the uric acid catabolism enzyme.
  • expression of the at least one gene encoding a transporter of uric acid is controlled by the same promoter that controls expression of the uric acid catabolism enzyme.
  • at least one gene encoding a transporter of uric acid and the uric acid catabolism enzyme are divergently transcribed from a promoter region.
  • each of genes encoding the at least one gene encoding a transporter of uric acid and the gene encoding the uric acid catabolism enzyme is controlled by different promoters.
  • the promoter is not operably linked with the at least one gene encoding a transporter of uric acid in nature.
  • the at least one gene encoding the transporter of uric acid is controlled by its native promoter.
  • the at least one gene encoding the transporter of uric acid is controlled by an inducible promoter.
  • the at least one gene encoding the transporter of uric acid is controlled by a promoter that is stronger than its native promoter.
  • the at least one gene encoding the transporter of uric acid is controlled by a constitutive promoter.
  • the promoter is an inducible promoter. Inducible promoters are described in more detail infra.
  • the at least one gene encoding a transporter of uric acid is located on a plasmid in the host cell, such as a bacterial cell. In another embodiment, the at least one gene encoding a transporter of uric acid is located in the chromosome of the host cell, such as a bacterial cell.
  • a native copy of the at least one gene encoding a transporter of uric acid is located in the chromosome of the host cell, such as a bacterial cell, and a copy of at least one gene encoding a transporter of uric acid from a different species of bacteria is located on a plasmid in the host cell, such as a bacterial cell.
  • a native copy of the at least one gene encoding a transporter of uric acid is located on a plasmid in the host cell, such as a bacterial cell, and a copy of at least one gene encoding a transporter of uric acid from a different species of bacteria is located on a plasmid in the host cell, such as a bacterial cell.
  • a native copy of the at least one gene encoding a transporter of uric acid is located in the chromosome of the host cell, such as a bacterial cell, and a copy of the at least one gene encoding a transporter of uric acid from a different species of bacteria is located in the chromosome of the host cell, such as a bacterial cell.
  • the at least one native gene encoding the transporter in the host cell, such as a bacterial cell is not modified, and one or more additional copies of the native transporter are inserted into the genome.
  • the one or more additional copies of the native transporter that is inserted into the genome are under the control of the same inducible promoter that controls expression of the gene encoding the uric acid catabolism enzyme, e.g., the FNR promoter, or a different inducible promoter than the one that controls expression of the uric acid catabolism enzyme, or a constitutive promoter.
  • the at least one native gene encoding the transporter is not modified, and one or more additional copies of the transporter from a different bacterial species is inserted into the genome of the host cell, such as a bacterial cell.
  • the one or more additional copies of the transporter inserted into the genome of the bacterial cell are under the control of the same inducible promoter that controls expression of the gene encoding the uric acid catabolism enzyme, e.g., the FNR promoter, or a different inducible promoter than the one that controls expression of the gene encoding the uric acid catabolism enzyme, or a constitutive promoter.
  • at least one native gene encoding the transporter in the genetically modified host cell such as a bacterial cell, is not modified, and one or more additional copies of at least one native gene encoding the transporter are present in the host cell, such as a bacterial cell, on a plasmid.
  • the at least one native gene encoding the transporter present in the host cell, such as a bacterial cell, on a plasmid is under the control of the same inducible promoter that controls expression of the gene encoding the uric acid catabolism enzyme, e.g., the FNR promoter, or a different inducible promoter than the one that controls expression of the gene encoding the uric acid catabolism enzyme, or a constitutive promoter.
  • the at least one native gene encoding the transporter is not modified, and a copy of at least one gene encoding the transporter from a different bacterial species is present in the host cell, such as a bacterial cell, on a plasmid.
  • the copy of at least one gene encoding the transporter from a different bacterial species is under the control of the same inducible promoter that controls expression of the gene encoding the uric acid catabolism enzyme, e.g., the FNR promoter, or a different inducible promoter than the one that controls expression of the gene encoding the uric acid catabolism enzyme, or a constitutive promoter.
  • the bacterium is E. coli Nissle, and the at least one native gene encoding the transporter in E. coli Nissle is not modified; one or more additional copies at least one native gene encoding the transporter from E. coli Nissle is inserted into the E.
  • the coli Nissle genome under the control of the same inducible promoter that controls expression of the gene encoding the uric acid catabolism enzyme, e.g., the FNR promoter, or a different inducible promoter than the one that controls expression of the gene encoding the uric acid catabolism enzyme, or a constitutive promoter.
  • the at least one native gene encoding the transporter in E. coli Nissle is not modified, and a copy of at least one gene encoding the transporter from a different bacterial species is inserted into the E.
  • the coli Nissle genome under the control of the same inducible promoter that controls expression of the gene encoding the uric acid catabolism enzyme, e.g., the FNR promoter, or a different inducible promoter than the one that controls expression of the gene encoding the uric acid catabolism enzyme, or a constitutive promoter.
  • the host cells when the transporter of uric acid is expressed in the recombinant host cells, such as the bacterial cells, import 10% more uric acid into the host cell, such as the bacterial cell, when the transporter is expressed than unmodified bacteria of the same bacterial subtype under the same conditions.
  • the host cells when the transporter of uric acid is expressed in the recombinant host cells, such as the bacterial cells, import 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more uric acid, into the host cell, such as the bacterial cell, when the transporter is expressed than unmodified host cell, such as the bacterial cell, of the same bacterial subtype under the same conditions.
  • the host cells when the transporter of uric acid is expressed in the recombinant host cells, such as bacterial cells, the host cells, such as bacterial cells, import two-fold more uric acid into the cell when the transporter is expressed than unmodified host cell, such as the bacterial cell, of the same bacterial subtype under the same conditions.
  • the host cells when the transporter of uric acid is expressed in the recombinant host cells, such as bacterial cells, the host cells, such as bacterial cells, import three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, or ten-fold more uric acid into the cell when the transporter is expressed than unmodified host cells, such as bacterial cells, of the same bacterial subtype under the same conditions.
  • the recombinant host cells, such as bacterial cells, described herein further comprise at least one uric acid transporter.
  • the recombinant host cells, such as bacterial cells, described herein comprise two uric acid transporters.
  • the recombinant host cells, such as bacterial cells, described herein comprise three uric acid transporters. In another embodiment, the recombinant host cells, such as bacterial cells, described herein comprise four uric acid transporters. In another embodiment, the recombinant host cells, such as bacterial cells, described herein comprise five uric acid transporters.
  • the transporter of urate imports urate into the bacterial cell. Multiple distinct transporters of uric acid are well known in the art and are described, below.
  • the at least one gene encoding the uric acid transporter is a gene encoding UacT.
  • the uacT gene has at least about 80% identity with the sequence of SEQ ID NO: 5. Accordingly, in one embodiment, the uacT gene has at least about 90% identity with the sequence of SEQ ID NO: 5. Accordingly, in one embodiment, the uacT gene has at least about 95% identity with the sequence of SEQ ID NO: 5. Accordingly, in one embodiment, the uacT gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 5. In another embodiment, the uacT gene comprises the sequence of SEQ ID NO: 5.
  • the uacT gene consists of the sequence of SEQ ID NO: 5. [0264] In one embodiment, the uacT gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 10. Accordingly, in one embodiment, the uacT gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 10. Accordingly, in one embodiment, the uacT gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 10.
  • the uacT gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 10.
  • the uacT gene encodes a protein comprising the sequence of SEQ ID NO: 10.
  • the uacT gene encodes a protein consisting of the sequence of SEQ ID NO: 10.
  • the at least one gene encoding the uric acid transporter is a gene encoding UacT.
  • the uacT gene has at least about 80% identity with the sequence of SEQ ID NO: 211. Accordingly, in one embodiment, the uacT gene has at least about 90% identity with the sequence of SEQ ID NO: 211. Accordingly, in one embodiment, the uacT gene has at least about 95% identity with the sequence of SEQ ID NO: 211. Accordingly, in one embodiment, the uacT gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 211.
  • the uacT gene comprises the sequence of SEQ ID NO: 211. In yet another embodiment the uacT gene consists of the sequence of SEQ ID NO: 211. [0266] In one embodiment, the uacT gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 220. Accordingly, in one embodiment, the uacT gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 220. Accordingly, in one embodiment, the uacT gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 220.
  • the uacT gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 220.
  • the uacT gene encodes a protein comprising the sequence of SEQ ID NO: 220.
  • the uacT gene encodes a protein consisting of the sequence of SEQ ID NO: 220.
  • the at least one gene encoding the uric acid transporter is a gene encoding UacT.
  • the uacT gene has at least about 80% identity with the sequence of SEQ ID NO: 212. Accordingly, in one embodiment, the uacT gene has at least about 90% identity with the sequence of SEQ ID NO: 212. Accordingly, in one embodiment, the uacT gene has at least about 95% identity with the sequence of SEQ ID NO: 212. Accordingly, in one embodiment, the uacT gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 212.
  • the uacT gene comprises the sequence of SEQ ID NO: 212. In yet another embodiment the uacT gene consists of the sequence of SEQ ID NO: 212. [0268] In one embodiment, the uacT gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 221. Accordingly, in one embodiment, the uacT gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 221. Accordingly, in one embodiment, the uacT gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 221.
  • the uacT gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 221.
  • the uacT gene encodes a protein comprising the sequence of SEQ ID NO: 221.
  • the uacT gene encodes a protein consisting of the sequence of SEQ ID NO: 221.
  • the at least one gene encoding the uric acid transporter is a gene encoding UacT.
  • the uacT gene has at least about 80% identity with the sequence of SEQ ID NO: 213. Accordingly, in one embodiment, the uacT gene has at least about 90% identity with the sequence of SEQ ID NO: 213. Accordingly, in one embodiment, the uacT gene has at least about 95% identity with the sequence of SEQ ID NO: 213. Accordingly, in one embodiment, the uacT gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 213. In another embodiment, the uacT gene comprises the sequence of SEQ ID NO: 213.
  • the uacT gene consists of the sequence of SEQ ID NO: 213. [0270] In one embodiment, the uacT gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 222. Accordingly, in one embodiment, the uacT gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 222. Accordingly, in one embodiment, the uacT gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 222.
  • the uacT gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 222.
  • the uacT gene encodes a protein comprising the sequence of SEQ ID NO: 222.
  • the uacT gene encodes a protein consisting of the sequence of SEQ ID NO: 222.
  • the transporter gene has at least about 80% identity with the sequence of SEQ ID NO: 234.
  • the transporter gene has at least about 90% identity with the sequence of SEQ ID NO: 234. Accordingly, in one embodiment, the transporter gene has at least about 95% identity with the sequence of SEQ ID NO: 234. Accordingly, in one embodiment, the transporter gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 234. In another embodiment, the transporter gene comprises the sequence of SEQ ID NO: 234. In yet another embodiment the transporter gene consists of the sequence of SEQ ID NO: 234.
  • the transporter gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 246. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 246. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 246. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 246.
  • the transporter gene encodes a protein comprising the sequence of SEQ ID NO: 246. In yet another embodiment the transporter gene encodes a protein consisting of the sequence of SEQ ID NO: 246. [0273] In one embodiment, the transporter gene has at least about 80% identity with the sequence of SEQ ID NO: 235. Accordingly, in one embodiment, the transporter gene has at least about 90% identity with the sequence of SEQ ID NO: 235. Accordingly, in one embodiment, the transporter gene has at least about 95% identity with the sequence of SEQ ID NO: 235.
  • the transporter gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 235.
  • the transporter gene comprises the sequence of SEQ ID NO: 235.
  • the transporter gene consists of the sequence of SEQ ID NO: 235.
  • the transporter gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 247. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 247.
  • the transporter gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 247. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 247. In another embodiment, the transporter gene encodes a protein comprising the sequence of SEQ ID NO: 247. In yet another embodiment the transporter gene encodes a protein consisting of the sequence of SEQ ID NO: 247.
  • the transporter gene has at least about 80% identity with the sequence of SEQ ID NO: 236. Accordingly, in one embodiment, the transporter gene has at least about 90% identity with the sequence of SEQ ID NO: 236. Accordingly, in one embodiment, the transporter gene has at least about 95% identity with the sequence of SEQ ID NO: 236. Accordingly, in one embodiment, the transporter gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 236. In another embodiment, the transporter gene comprises the sequence of SEQ ID NO: 236.
  • the transporter gene consists of the sequence of SEQ ID NO: 236. [0276] In one embodiment, the transporter gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 248. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 248. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 248.
  • the transporter gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 248.
  • the transporter gene encodes a protein comprising the sequence of SEQ ID NO: 248.
  • the transporter gene encodes a protein consisting of the sequence of SEQ ID NO: 248.
  • the transporter gene has at least about 80% identity with the sequence of SEQ ID NO: 237. Accordingly, in one embodiment, the transporter gene has at least about 90% identity with the sequence of SEQ ID NO: 237.
  • the transporter gene has at least about 95% identity with the sequence of SEQ ID NO: 237. Accordingly, in one embodiment, the transporter gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 237. In another embodiment, the transporter gene comprises the sequence of SEQ ID NO: 237. In yet another embodiment the transporter gene consists of the sequence of SEQ ID NO: 237. [0278] In one embodiment, the transporter gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 249.
  • the transporter gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 249. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 249. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 249. In another embodiment, the transporter gene encodes a protein comprising the sequence of SEQ ID NO: 249.
  • the transporter gene encodes a protein consisting of the sequence of SEQ ID NO: 249.
  • the transporter gene has at least about 80% identity with the sequence of SEQ ID NO: 238. Accordingly, in one embodiment, the transporter gene has at least about 90% identity with the sequence of SEQ ID NO: 238. Accordingly, in one embodiment, the transporter gene has at least about 95% identity with the sequence of SEQ ID NO: 238. Accordingly, in one embodiment, the transporter gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 238.
  • the transporter gene comprises the sequence of SEQ ID NO: 238. In yet another embodiment the transporter gene consists of the sequence of SEQ ID NO: 238. [0280] In one embodiment, the transporter gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 250. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 250. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 250.
  • the transporter gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 250.
  • the transporter gene encodes a protein comprising the sequence of SEQ ID NO: 250.
  • the transporter gene encodes a protein consisting of the sequence of SEQ ID NO: 250.
  • the transporter gene has at least about 80% identity with the sequence of SEQ ID NO: 239. Accordingly, in one embodiment, the transporter gene has at least about 90% identity with the sequence of SEQ ID NO: 239.
  • the transporter gene has at least about 95% identity with the sequence of SEQ ID NO: 239. Accordingly, in one embodiment, the transporter gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 239. In another embodiment, the transporter gene comprises the sequence of SEQ ID NO: 239. In yet another embodiment the transporter gene consists of the sequence of SEQ ID NO: 239. [0282] In one embodiment, the transporter gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 251.
  • the transporter gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 251. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 251. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 251. In another embodiment, the transporter gene encodes a protein comprising the sequence of SEQ ID NO: 251.
  • the transporter gene encodes a protein consisting of the sequence of SEQ ID NO: 251.
  • the transporter gene has at least about 80% identity with the sequence of SEQ ID NO: 240. Accordingly, in one embodiment, the transporter gene has at least about 90% identity with the sequence of SEQ ID NO: 240. Accordingly, in one embodiment, the transporter gene has at least about 95% identity with the sequence of SEQ ID NO: 240. Accordingly, in one embodiment, the transporter gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 240.
  • the transporter gene comprises the sequence of SEQ ID NO: 240. In yet another embodiment the transporter gene consists of the sequence of SEQ ID NO: 240. [0284] In one embodiment, the transporter gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 252. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 252. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 252.
  • the transporter gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 252.
  • the transporter gene encodes a protein comprising the sequence of SEQ ID NO: 252.
  • the transporter gene encodes a protein consisting of the sequence of SEQ ID NO: 252.
  • the transporter gene has at least about 80% identity with the sequence of SEQ ID NO: 241. Accordingly, in one embodiment, the transporter gene has at least about 90% identity with the sequence of SEQ ID NO: 241.
  • the transporter gene has at least about 95% identity with the sequence of SEQ ID NO: 241. Accordingly, in one embodiment, the transporter gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 241. In another embodiment, the transporter gene comprises the sequence of SEQ ID NO: 241. In yet another embodiment the transporter gene consists of the sequence of SEQ ID NO: 241. [0286] In one embodiment, the transporter gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 253.
  • the transporter gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 253. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 253. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 253. In another embodiment, the transporter gene encodes a protein comprising the sequence of SEQ ID NO: 253.
  • the transporter gene encodes a protein consisting of the sequence of SEQ ID NO: 253.
  • the transporter gene has at least about 80% identity with the sequence of SEQ ID NO: 242. Accordingly, in one embodiment, the transporter gene has at least about 90% identity with the sequence of SEQ ID NO: 242. Accordingly, in one embodiment, the transporter gene has at least about 95% identity with the sequence of SEQ ID NO: 242. Accordingly, in one embodiment, the transporter gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 242.
  • the transporter gene comprises the sequence of SEQ ID NO: 242. In yet another embodiment the transporter gene consists of the sequence of SEQ ID NO: 242. [0288] In one embodiment, the transporter gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 254. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 254. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 254.
  • the transporter gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 254.
  • the transporter gene encodes a protein comprising the sequence of SEQ ID NO: 254.
  • the transporter gene encodes a protein consisting of the sequence of SEQ ID NO: 254.
  • the transporter gene has at least about 80% identity with the sequence of SEQ ID NO: 243. Accordingly, in one embodiment, the transporter gene has at least about 90% identity with the sequence of SEQ ID NO: 243.
  • the transporter gene has at least about 95% identity with the sequence of SEQ ID NO: 243. Accordingly, in one embodiment, the transporter gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 243. In another embodiment, the transporter gene comprises the sequence of SEQ ID NO: 243. In yet another embodiment the transporter gene consists of the sequence of SEQ ID NO: 243. [0290] In one embodiment, the transporter gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 255.
  • the transporter gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 255. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 255. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 255. In another embodiment, the transporter gene encodes a protein comprising the sequence of SEQ ID NO: 255.
  • the transporter gene encodes a protein consisting of the sequence of SEQ ID NO: 255.
  • the transporter gene has at least about 80% identity with the sequence of SEQ ID NO: 244. Accordingly, in one embodiment, the transporter gene has at least about 90% identity with the sequence of SEQ ID NO: 244. Accordingly, in one embodiment, the transporter gene has at least about 95% identity with the sequence of SEQ ID NO: 244. Accordingly, in one embodiment, the transporter gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 244.
  • the transporter gene comprises the sequence of SEQ ID NO: 244. In yet another embodiment the transporter gene consists of the sequence of SEQ ID NO: 244. [0292] In one embodiment, the transporter gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 256. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 256. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 256.
  • the transporter gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 256.
  • the transporter gene encodes a protein comprising the sequence of SEQ ID NO: 256.
  • the transporter gene encodes a protein consisting of the sequence of SEQ ID NO: 256.
  • the transporter gene has at least about 80% identity with the sequence of SEQ ID NO: 245. Accordingly, in one embodiment, the transporter gene has at least about 90% identity with the sequence of SEQ ID NO: 245.
  • the transporter gene has at least about 95% identity with the sequence of SEQ ID NO: 245. Accordingly, in one embodiment, the transporter gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 245. In another embodiment, the transporter gene comprises the sequence of SEQ ID NO: 245. In yet another embodiment the transporter gene consists of the sequence of SEQ ID NO: 245. [0294] In one embodiment, the transporter gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 257.
  • the transporter gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 257. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 257. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 257. In another embodiment, the transporter gene encodes a protein comprising the sequence of SEQ ID NO: 257.
  • the transporter gene encodes a protein consisting of the sequence of SEQ ID NO: 257.
  • the transporter gene has at least about 80% identity with the sequence of SEQ ID NO: 258. Accordingly, in one embodiment, the transporter gene has at least about 90% identity with the sequence of SEQ ID NO: 258. Accordingly, in one embodiment, the transporter gene has at least about 95% identity with the sequence of SEQ ID NO: 258. Accordingly, in one embodiment, the transporter gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 258.
  • the transporter gene comprises the sequence of SEQ ID NO: 258. In yet another embodiment the transporter gene consists of the sequence of SEQ ID NO: 258. [0296] In one embodiment, the transporter gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 259. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 259. Accordingly, in one embodiment, the transporter gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 259.
  • the transporter gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 259.
  • the transporter gene encodes a protein comprising the sequence of SEQ ID NO: 259.
  • the transporter gene encodes a protein consisting of the sequence of SEQ ID NO: 259.
  • the importer gene encodes a polypeptide with a F275M mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 10.
  • the importer gene encodes a polypeptide with a A246S mutation referenced by the polypeptide encoded by the gene sequence having the sequence of SEQ ID NO: 10.
  • the at least one gene encoding the uric acid transporter is a gene encoding YgfU, or the ygfu gene.
  • YgfU is a uric acid specific proton symporter, a member of the ubiquitous nucleobase-ascorbate transporter family (NCS2).
  • the ygfU gene has at least about 80% identity with the sequence of SEQ ID NO: 231.
  • the ygfU gene has at least about 90% identity with the sequence of SEQ ID NO: 231. Accordingly, in one embodiment, the ygfU gene has at least about 95% identity with the sequence of SEQ ID NO: 231. Accordingly, in one embodiment, the ygfU gene has at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 231. In another embodiment, the ygfU gene comprises the sequence of SEQ ID NO: 231. In yet another embodiment the ygfU gene consists of the sequence of SEQ ID NO: 231.
  • the recombinant bacterial cells described herein comprise a heterologous gene sequence encoding one or more uric acid catabolism enzyme, and a heterologous gene encoding a urate importer.
  • the recombinant bacterial cells comprise an aegA gene, and a ygfU gene.
  • the recombinant bacterial cells comprise an ygfT gene, and a ygfU gene.
  • the recombinant bacterial cells comprise an aegA gene, an ygfT gene, and a ygfU gene.
  • the ygfU gene encodes a protein having at least about 80% identity with the sequence of SEQ ID NO: 201. Accordingly, in one embodiment, the ygfU gene encodes a protein having at least about 90% identity with the sequence of SEQ ID NO: 201. Accordingly, in one embodiment, the ygfU gene encodes a protein having at least about 95% identity with the sequence of SEQ ID NO: 201.
  • the ygfU gene encodes a protein having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 201.
  • the ygfU gene encodes a protein comprising the sequence of SEQ ID NO: 201.
  • the ygfU gene encodes a protein consisting of the sequence of SEQ ID NO: 201. C.
  • the host cell such as a bacterial cell, comprises a stably maintained plasmid or chromosome carrying the gene(s) encoding the uricase and/or the importer(s), such that the uricase and/or the importer(s) can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo, e.g., in the gut.
  • the host cell such as a bacterial cell, comprises two or more distinct uricase and/or importer genes or operons.
  • the host cell such as a bacterial cell, comprises three or more distinct uricase and/or importer genes or operons. In some embodiments, the host cell, such as a bacterial host cell, comprises 4, 5, 6, 7, 8, 9, 10, or more distinct uricase and/or importer genes or operons. [0301] In some embodiments, the host cell, such as a bacterium, comprises multiple copies of the same uricase and/or importer genes. In some embodiments, the gene encoding the uricase and/or importer is present on a plasmid and operably linked to a directly or indirectly inducible promoter.
  • the gene encoding the uricase and/or importer is present on a plasmid and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions. In some embodiments, the gene encoding the uricase and/or importer is present on a chromosome and operably linked to a directly or indirectly inducible promoter. In some embodiments, the gene encoding the uricase and/or importer is present in the chromosome and operably linked to a promoter that is induced under low-oxygen or anaerobic conditions.
  • the gene encoding the uricase and/or importer is present on a plasmid and operably linked to a promoter that is induced by exposure to tetracycline, arabinose or Isopropyl ß-D-1-thiogalactopyranoside (IPTG).
  • the inducible promoter is a IPTG inducible promoter, e.g., Ptac.
  • the IPTG inducible promoter comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 1108.
  • the recombinant bacterium further comprises a gene sequence encoding a gene sequence encoding a transcriptional regulator, e.g., a repressor IPTG inducible promoter.
  • the gene sequence encoding a repressor comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 1105.
  • the repressor comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to, comprises, or consists of SEQ ID NO: 1106.
  • the host cells such as bacterial cells, comprise endogenous gene(s) encoding the IPTG sensing transcriptional regulator, LacI.
  • the lacI gene is heterologous.
  • the gene encoding the IPTG level-sensing transcriptional regulator, e.g., LacI is present on a plasmid.
  • the gene encoding the IPTG level-sensing transcriptional regulator, e.g., LacI, and the gene encoding the uricase or importer are present on different plasmids.
  • the gene encoding the IPTG level-sensing transcriptional regulator, e.g., LacI, and the gene encoding the uricase or importer are present on the same plasmid. In some embodiments, the gene encoding the IPTG level-sensing transcriptional regulator, e.g., LacI, is present on a chromosome. In some embodiments, the gene encoding the IPTG level-sensing transcriptional regulator, e.g., LacI, and the gene encoding the uricase or importer are present on different chromosomes.
  • the gene encoding the IPTG level-sensing transcriptional regulator, e.g., LacI, and the gene encoding the uricase or importer are present on the same chromosome, either at the same or a different insertion site.
  • expression of the transcriptional regulator is controlled by a different promoter than the promoter that controls expression of the gene encoding the uricase or importer, e.g., a constitutive promoter.
  • expression of the transcriptional regulator is controlled by the same promoter that controls expression of the uricase or importer.
  • the transcriptional regulator and the uricase or importer are divergently transcribed from a promoter region.
  • the promoter that is operably linked to the gene encoding the uricase or importer is directly induced by exogenous environmental conditions. In some embodiments, the promoter that is operably linked to the gene encoding the uricase or importer is indirectly induced by exogenous environmental conditions. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by exogenous environmental conditions specific to the small intestine of a mammal. In some embodiments, the promoter is directly or indirectly induced by low-oxygen or anaerobic conditions such as the environment of the mammalian gut.
  • the promoter is directly or indirectly induced by molecules or metabolites that are specific to the gut of a mammal. In some embodiments, the promoter is directly or indirectly induced by a molecule that is co-administered with the bacterial cell.
  • the inducible promoter is an anhydrotetracycline (ATC)-inducible promoter. In one embodiment, the inducible promoter is an IPTG promoter. In one embodiment, the IPTG promoter is Ptac. [0305] As used herein the term “pTac” or “pTac promoter” includes the minimal promoter having - 35 and -10 regions and at least the lac operator region.
  • the term “pTac” or “pTac promoter” may also include an RBS in addition the minimal promoter and the Lac operator region.
  • suitable RBSs are listed herein.
  • pTac promoter sequence comprises SEQ ID NO: 1108.
  • an RBS may be included at the 3’ end of SEQ ID NO: 1108.
  • the RBS comprises SEQ ID NO: 1107.
  • the bacterial cell comprises a gene encoding a uricase and/or importer expressed under the control of a fumarate and nitrate reductase regulator (FNR) responsive promoter.
  • FNR fumarate and nitrate reductase regulator
  • FNR is a major transcriptional activator that controls the switch from aerobic to anaerobic metabolism (Unden et al., 1997). In the anaerobic state, FNR dimerizes into an active DNA binding protein that activates hundreds of genes responsible for adapting to anaerobic growth. In the aerobic state, FNR is prevented from dimerizing by oxygen and is inactive.
  • FNR responsive promoters include, but are not limited to, the FNR responsive promoters listed in the chart, below. Underlined sequences are predicted ribosome binding sites, and bolded sequences are restriction sites used for cloning. Table 4: FNR Responsive Promoters [0307] In one embodiment, the FNR responsive promoter comprises SEQ ID NO: 43.
  • the FNR responsive promoter comprises SEQ ID NO: 44. In another embodiment, the FNR responsive promoter comprises SEQ ID NO: 45. In another embodiment, the FNR responsive promoter comprises SEQ ID NO: 46. In yet another embodiment, the FNR responsive promoter comprises SEQ ID NO: 47. [0308] In some embodiments, multiple distinct FNR nucleic acid sequences are inserted in the genetically engineered bacteria.
  • the genetically engineered bacteria comprise a gene encoding a uric acid catabolism enzyme and/or a gene encoding a urate importer expressed under the control of an alternate oxygen level-dependent promoter, e.g., DNR (Trunk et al., 2010) or ANR (Ray et al., 1997).
  • an alternate oxygen level-dependent promoter e.g., DNR (Trunk et al., 2010) or ANR (Ray et al., 1997.
  • expression of the uric acid catabolism enzyme gene or urate importer gene is particularly activated in a low-oxygen or anaerobic environment, such as in the gut.
  • the mammalian gut is a human mammalian gut.
  • the bacterial cell comprises an oxygen-level dependent transcriptional regulator, e.g., FNR, ANR, or DNR, and corresponding promoter from a different bacterial species.
  • the heterologous oxygen-level dependent transcriptional regulator and promoter increase the transcription of genes operably linked to said promoter, e.g., the gene encoding the uric acid catabolism enzyme or the gene encoding a urate importer, in a low-oxygen or anaerobic environment, as compared to the native gene(s) and promoter in the bacteria under the same conditions.
  • the non-native oxygen-level dependent transcriptional regulator is an FNR protein from N.
  • the corresponding wild-type transcriptional regulator is left intact and retains wild-type activity. In alternate embodiments, the corresponding wild-type transcriptional regulator is deleted or mutated to reduce or eliminate wild- type activity.
  • the genetically engineered bacteria comprise a wild-type oxygen- level dependent transcriptional regulator, e.g., FNR, ANR, or DNR, and corresponding promoter that is mutated relative to the wild-type promoter from bacteria of the same subtype.
  • the mutated promoter enhances binding to the wild-type transcriptional regulator and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the uric acid catabolism enzyme or the gene encoding the urate importer, in a low-oxygen or anaerobic environment, as compared to the wild-type promoter under the same conditions.
  • the genetically engineered bacteria comprise a wild-type oxygen-level dependent promoter, e.g., FNR, ANR, or DNR promoter, and corresponding transcriptional regulator that is mutated relative to the wild-type transcriptional regulator from bacteria of the same subtype.
  • the mutated transcriptional regulator enhances binding to the wild-type promoter and increases the transcription of genes operably linked to said promoter, e.g., the gene encoding the uric acid catabolism enzyme or the gene encoding the urate importer, in a low-oxygen or anaerobic environment, as compared to the wild-type transcriptional regulator under the same conditions.
  • the mutant oxygen-level dependent transcriptional regulator is an FNR protein comprising amino acid substitutions that enhance dimerization and FNR activity (see, e.g., Moore et al., (2006).
  • the bacterial cells comprise multiple copies of the endogenous gene encoding the oxygen level-sensing transcriptional regulator, e.g., the FNR gene.
  • the gene encoding the oxygen level-sensing transcriptional regulator is present on a plasmid.
  • the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the uric acid catabolism enzyme or the gene encoding the urate importer are present on different plasmids.
  • the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the uric acid catabolism enzyme or the gene encoding the urate importer are present on the same plasmid. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator is present on a chromosome. In some embodiments, the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the uric acid catabolism enzyme or the gene encoding the urate importer are present on different chromosomes.
  • the gene encoding the oxygen level-sensing transcriptional regulator and the gene encoding the uric acid catabolism enzyme or the gene encoding the urate importer are present on the same chromosome. In some instances, it may be advantageous to express the oxygen level-sensing transcriptional regulator under the control of an inducible promoter in order to enhance expression stability. In some embodiments, expression of the transcriptional regulator is controlled by a different promoter than the promoter that controls expression of the gene encoding the uric acid catabolism enzyme or the gene encoding the urate importer.
  • expression of the transcriptional regulator is controlled by the same promoter that controls expression of the uric acid catabolism enzyme or the gene encoding the urate importer.
  • the transcriptional regulator and the uric acid catabolism enzyme are divergently transcribed from a promoter region.
  • any of the gene(s) of the present disclosure may be integrated into the chromosome of a host cell, such as a bacterial chromosome, at one or more integration sites.
  • one or more copies of one or more gene(s) encoding a uric acid catabolism enzyme or urate importer may be integrated into the chromosome of a host cell, such as a bacterial chromosome.
  • Having multiple copies of the gene or gene(s) integrated into the chromosome allows for greater production of the uric acid catabolism enzyme(s) and also permits fine-tuning of the level of expression.
  • different circuits described herein, in addition to the therapeutic gene(s) or gene cassette(s) could be integrated into the chromosome of a host cell, such as a bacterial chromosome, at one or more different integration sites to perform multiple different functions.
  • the genetically engineered bacteria comprise a stably maintained plasmid or chromosome carrying a gene for producing a uric acid catabolism enzyme, such that the uric acid catabolism enzyme can be expressed in the host cell, and the host cell is capable of survival and/or growth in vitro, e.g., in medium, and/or in vivo.
  • a bacterium may comprise multiple copies of the gene encoding the uric acid catabolism enzyme.
  • the gene encoding the uric acid catabolism enzyme is expressed on a low-copy plasmid. In some embodiments, the low-copy plasmid may be useful for increasing stability of expression.
  • the low-copy plasmid may be useful for decreasing leaky expression under non-inducing conditions.
  • the gene encoding the uric acid catabolism enzyme is expressed on a high-copy plasmid.
  • the high-copy plasmid may be useful for increasing expression of the uric acid catabolism enzyme.
  • the gene encoding the uric acid catabolism enzyme is expressed on a chromosome.
  • the bacteria are genetically engineered to include multiple mechanisms of action (MOAs), e.g., circuits producing multiple copies of the same product (e.g., to enhance copy number) or circuits performing multiple different functions.
  • MOAs mechanisms of action
  • the genetically engineered bacteria may include four copies of the gene encoding a particular uric acid catabolism enzyme inserted at four different insertion sites.
  • the genetically engineered bacteria may include three copies of the gene encoding a particular uric acid catabolism enzyme inserted at three different insertion sites and three copies of the gene encoding a different uric acid catabolism enzyme inserted at three different insertion sites.
  • the genetically engineered host cells such as genetically engineered bacteria, of the disclosure produce at least about 1.5-fold, at least about 2-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 30-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600- fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1,000-fold, or at least about 1,500-fold more of the uric acid catabolism enzyme, and/or transcript of the gene(s) in the operon as compared to unmodified host cells, such as unmodified bacteria, of the same subtype under the same conditions.
  • qPCR quantitative PCR
  • Primers specific for uric acid catabolism enzyme the gene(s) may be designed and used to detect mRNA in a sample according to methods known in the art.
  • a fluorophore is added to a sample reaction mixture that may contain uric acid catabolism enzyme mRNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. The reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods.
  • the heating and cooling is repeated for a predetermined number of cycles.
  • the reaction mixture is heated and cooled to 90-100° C, 60-70° C, and 30-50° C for a predetermined number of cycles.
  • the reaction mixture is heated and cooled to 93-97° C, 55-65° C, and 35-45° C for a predetermined number of cycles.
  • the accumulating amplicon is quantified after each cycle of the qPCR.
  • the number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT). At least one CT result for each sample is generated, and the CT result(s) may be used to determine mRNA expression levels of the uric acid catabolism enzyme gene(s).
  • qPCR quantitative PCR
  • Primers specific for uric acid catabolism enzyme the gene(s) may be designed and used to detect mRNA in a sample according to methods known in the art.
  • a fluorophore is added to a sample reaction mixture that may contain uric acid catabolism enzyme mRNA, and a thermal cycler is used to illuminate the sample reaction mixture with a specific wavelength of light and detect the subsequent emission by the fluorophore. The reaction mixture is heated and cooled to predetermined temperatures for predetermined time periods.
  • the heating and cooling is repeated for a predetermined number of cycles.
  • the reaction mixture is heated and cooled to 90-100° C, 60-70° C, and 30-50° C for a predetermined number of cycles.
  • the reaction mixture is heated and cooled to 93-97° C, 55-65° C, and 35-45° C for a predetermined number of cycles.
  • the accumulating amplicon is quantified after each cycle of the qPCR.
  • the number of cycles at which fluorescence exceeds the threshold is the threshold cycle (CT). At least one CT result for each sample is generated, and the CT result(s) may be used to determine mRNA expression levels of the uric acid catabolism enzyme gene(s).
  • thermoregulators may be advantageous because of strong transcriptional control without the use of external chemicals or specialized media.
  • Thermoregulated protein expression using the mutant cI857 repressor and the pL and/or pR phage ⁇ promoters have been used to engineer recombinant bacterial strains.
  • a gene of interest cloned downstream of the ⁇ promoters can be efficiently regulated by the mutant thermolabile cI857 repressor of bacteriophage ⁇ .
  • cI857 binds to the oL or regions of the pR promoter and inhibits transcription by RNA polymerase.
  • the functional cI857 dimer is destabilized, binding to the oL or DNA sequences is abrogated, and mRNA transcription is initiated.
  • thermoregulated promoter may be induced in culture, e.g., grown in a flask, fermenter or other appropriate culture vessel, e.g., used during cell growth, cell expansion, fermentation, recovery, purification, formulation, and/or manufacture.
  • Bacteria comprising gene sequences or gene cassettes either indirectly or directly operably linked to a temperature sensitive system or promoter may, for example, could be induced by temperatures between 37°C and 42°C.
  • the cultures may be grown aerobically. Alternatively, the cultures are grown anaerobically.
  • the host cell such as a bacterial host cell, described herein comprise one or more gene sequence(s) or gene cassette(s) which are directly or indirectly operably linked to a temperature regulated promoter.
  • the gene sequence(s) or gene cassette(s) are induced in vitro during growth, preparation, or manufacturing of the strain prior to in vivo administration.
  • the gene sequence(s) are induced upon or during in vivo administration.
  • the gene sequence(s) are induced during in vitro growth, preparation, or manufacturing of the strain prior to in vivo administration and upon or during in vivo administration.
  • the genetically engineered host cell such as a bacterial host cell, further comprise gene sequence (s) encoding a transcription factor which is capable of binding to the temperature sensitive promoter.
  • the transcription factor is a repressor of transcription.
  • the thermoregulated promoter is operably linked to a construct having gene sequence(s) or gene cassette(s) encoding one or more protein(s) of interest jointly with a second promoter, e.g., a second constitutive or inducible promoter.
  • two promoters are positioned proximally to the construct and drive its expression, wherein the thermoregulated promoter is induced under a first set of exogenous conditions, and the second promoter is induced under a second set of exogenous conditions.
  • the first and second conditions may be two sequential culture conditions (i.e., during preparation of the culture in a flask, fermenter or other appropriate culture vessel, e.g., thermoregulation and arabinose or IPTG).
  • the first inducing conditions may be culture conditions, e.g., permissive temperature
  • the second inducing conditions may be in vivo conditions.
  • thermoregulated promoters drive expression of one or more protein(s) of interest in combination with an oxygen regulated promoter, e.g., FNR, driving the expression of the same gene sequence(s).
  • an oxygen regulated promoter e.g., FNR
  • the thermoregulated promoter drives the expression of one or more protein(s) of interest from a low-copy plasmid or a high copy plasmid or a biosafety system plasmid described herein.
  • the thermoregulated promoter drives the expression of one or more protein(s) of interest from a construct which is integrated into the host cell chromosome, such as a bacterial chromosome. Exemplary insertion sites are described herein.
  • the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 313.
  • the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 316.
  • the thermoregulated construct further comprises a gene encoding mutant cI857 repressor, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest.
  • the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 233.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 312.
  • the thermoregulated construct further comprises a gene encoding mutant cI38 repressor, which is divergently transcribed from the same promoter as the one or more one or more protein(s) of interest.
  • the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with any of the sequences of SEQ ID NO: 314.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 315.
  • the genetically engineered bacteria comprise one or more gene sequence(s) encoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity with the polypeptide encoded by any of the sequences of SEQ ID NO: 316.
  • SEQ ID NOs: 233, and 312-316 are shown in Table 5.
  • the bacterial cells comprise gene(s) encoding a temperature sensing transcriptional regulator/repressor described herein, e.g., cI857 or a mutant thereof.
  • the gene encoding the temperature sensing transcriptional regulator is present on a plasmid.
  • the gene encoding the temperature sensing transcriptional regulator, and the gene encoding the uricase/uric acid transporter are present on different plasmids.
  • the gene encoding the temperature sensing transcriptional regulator, and the gene encoding the uricase or uric acid transporter are present on the same plasmid.
  • the gene encoding the temperature sensing transcriptional regulator is present on a chromosome. In some embodiments, the gene encoding the temperature sensing transcriptional regulator, and the gene encoding the uricase or uric acid transporter are present on different chromosomes. In some embodiments, the gene encoding the temperature sensing transcriptional regulator, and the gene encoding the uricase or uric acid transporter are present on the same chromosome, either at the same or at different insertion sites. In some embodiments, expression of temperature sensing transcriptional regulator is controlled by a different promoter than the promoter that controls expression of the gene encoding the uricase or uric acid transporter, e.g., a constitutive promoter.
  • the recombinant bacterial cell comprises one or more E. coli Nissle bacteriophage, e.g., Phage 1, Phage 2, and Phage 3.
  • the genetically recombinant bacterial cell comprises one or mutations in Phage 3. Such mutations include deletions, insertions, substitutions and inversions and are located in or encompass one or more Phage 3 genes.
  • the one or more insertions comprise an antibiotic cassette.
  • the mutation is a deletion.
  • the genetically engineered bacteria comprise one or more deletions, which are located in or comprise one or more genes selected from ECOLIN_09965, ECOLIN_09970, ECOLIN_09975, ECOLIN_09980, ECOLIN_09985, ECOLIN_09990, ECOLIN_09995, ECOLIN_10000, ECOLIN_10005, ECOLIN_10010, ECOLIN_10015, ECOLIN_10020, ECOLIN_10025, ECOLIN_10030, ECOLIN_10035, ECOLIN_10040, ECOLIN_10045, ECOLIN_10050, ECOLIN_10055, ECOLIN_10065, ECOLIN_10070, ECOLIN_10075, ECOLIN_10080, ECOLIN_10085, ECOLIN_10090, ECOLIN_10095, ECOLIN_10100, ECOLIN_10105
  • the genetically engineered bacteria comprise a complete or partial deletion of one or more of ECOLIN_10110, ECOLIN_10115, ECOLIN_10120, ECOLIN_10125, ECOLIN_10130, ECOLIN_10135, ECOLIN_10140, ECOLIN_10145, ECOLIN_10150, ECOLIN_10160, ECOLIN_10165, ECOLIN_10170, and ECOLIN_10175.
  • the deletion is a complete deletion of ECOLIN_10110, ECOLIN_10115, ECOLIN_10120, ECOLIN_10125, ECOLIN_10130, ECOLIN_10135, ECOLIN_10140, ECOLIN_10145, ECOLIN_10150, ECOLIN_10160, ECOLIN_10165, and ECOLIN_10170, and a partial deletion of ECOLIN_10175.
  • the sequence of SEQ ID NO: 1064 is deleted from the Phage 3 genome.
  • a sequence comprising SEQ ID NO: 1064 is deleted from the Phage 3 genome.
  • the recombinant bacterial cell comprises a modified pks island (colibactin island).
  • the recombinant bacterial cell comprises a modified clb sequence selected from one or more of the clbA, clbB, clbC, clbD, clbE, clbF, clbG, clbH, clbI, clbJ, clbK, clbL, clbM, clbN, clbO, clbP, clbQ, clbR, and clbS gene sequences, as compared to a suitable control, e.g., the native pks island in an unmodified bacterium of the same strain and/or subtype.
  • the modified clb sequence is an insertion, a substitution, and/or a deletion as compared to the control.
  • the modified clb sequence is a deletion of the clb island, e.g., clbA, clbB, clbC, clbD, clbE, clbF, clbG, clbH, clbI, clbJ, clbK, clbL, clbM, clbN, clbO, clbP, clbQ, clbR, and clbS.
  • the colibactin deletion is the whole island except for the clbS gene, e.g., a deletion of clbA, clbB, clbC, clbD, clbE, clbF, clbG, clbH, clbI, clbJ, clbK, clbL, clbM, clbN, clbO, clbP, clbQ, and clbR.
  • the clbS gene e.g., a deletion of clbA, clbB, clbC, clbD, clbE, clbF, clbG, clbH, clbI, clbJ, clbK, clbL, clbM, clbN, clbO, clbP
  • the modified endogenous colibactin island comprises one or more modified clb sequences selected from clbA (SEQ ID NO: 1065), clbB (SEQ ID NO: 1066), clbC (SEQ ID NO: 1067), clbD (SEQ ID NO: 1068), clbE (SEQ ID NO: 1069), clbF (SEQ ID NO: 1070), clbG (SEQ ID NO: 1071), clbH (SEQ ID NO: 1072), clbI (SEQ ID NO: 1073), clbJ (SEQ ID NO: 1074), clbK (SEQ ID NO: 1075), clbL (SEQ ID NO: 1076), clbM (SEQ ID NO: 1077), clbN (SEQ ID NO: 1078), clbO (SEQ ID NO: 1079), clbP (SEQ ID NO: 1065), clbB (
  • the modified endogenous colibactin island comprises a deletion of clbA (SEQ ID NO: 1065), clbB (SEQ ID NO: 1066), clbC (SEQ ID NO: 1067), clbD (SEQ ID NO: 1068), clbE (SEQ ID NO: 1069), clbF (SEQ ID NO: 1070), clbG (SEQ ID NO: 1071), clbH (SEQ ID NO: 1072), clbI (SEQ ID NO: 1073), clbJ (SEQ ID NO: 1074), clbK (SEQ ID NO: 1075), clbL (SEQ ID NO: 1076), clbM (SEQ ID NO: 1077), clbN (SEQ ID NO: 1078), clbO (SEQ ID NO: 1079), clbP (SEQ ID NO: 1080), clbQ (SEQ ID NO: 1065),
  • essential gene refers to a gene which is necessary to for cell growth and/or survival.
  • Bacterial essential genes are well known to one of ordinary skill in the art, and can be identified by directed deletion of genes and/or random mutagenesis and screening (see, for example, Zhang and Lin, 2009, DEG 5.0, a database of essential genes in both prokaryotes and eukaryotes, Nucl. Acids Res., 37: D455-D458 and Gerdes et al., Essential genes on metabolic maps, Curr. Opin. Biotechnol., 17(5):448-456, the entire contents of each of which are expressly incorporated herein by reference).
  • An “essential gene” may be dependent on the circumstances and environment in which an organism lives. For example, a mutation of, modification of, or excision of an essential gene may result in the recombinant bacteria of the disclosure becoming an auxotroph.
  • An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient.
  • An auxotrophic modification is intended to cause bacteria to die in the absence of an exogenously added nutrient essential for survival or growth because they lack the gene(s) necessary to produce that essential nutrient.
  • any of the genetically engineered bacteria described herein also comprise a deletion or mutation in a gene required for cell survival and/or growth.
  • the essential gene is an oligonucleotide synthesis gene, for example, thyA.
  • the essential gene is a cell wall synthesis gene, for example, dapA.
  • the essential gene is an amino acid gene, for example, serA or metA.
  • Any gene required for cell survival and/or growth may be targeted, including but not limited to, cysE, glnA, ilvD, leuB, lysA, serA, metA, glyA, hisB, ilvA, pheA, proA, thrC, trpC, tyrA, thyA, uraA, dapA, dapB, dapD, dapE, dapF, flhD, metB, metC, proAB, and thi1, as long as the corresponding wild-type gene product is not produced in the bacteria.
  • thymine is a nucleic acid that is required for bacterial cell growth; in its absence, bacteria undergo cell death.
  • the thyA gene encodes thimidylate synthetase, an enzyme that catalyzes the first step in thymine synthesis by converting dUMP to dTMP (Sat et al., 2003).
  • the bacterial cell of the disclosure is a thyA auxotroph in which the thyA gene is deleted and/or replaced with an unrelated gene.
  • a thyA auxotroph can grow only when sufficient amounts of thymine are present, e.g., by adding thymine to growth media in vitro, or in the presence of high thymine levels found naturally in the human gut in vivo.
  • the bacterial cell of the disclosure is auxotrophic in a gene that is complemented when the bacterium is present in the mammalian gut. Without sufficient amounts of thymine, the thyA auxotroph dies. In some embodiments, the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).
  • DAP Diaminopimelic acid
  • any of the genetically engineered bacteria described herein is a dapD auxotroph in which dapD is deleted and/or replaced with an unrelated gene.
  • a dapD auxotroph can grow only when sufficient amounts of DAP are present, e.g., by adding DAP to growth media in vitro. Without sufficient amounts of DAP, the dapD auxotroph dies.
  • the auxotrophic modification is used to ensure that the bacterial cell does not survive in the absence of the auxotrophic gene product (e.g., outside of the gut).
  • the genetically engineered bacterium of the present disclosure is a uraA auxotroph in which uraA is deleted and/or replaced with an unrelated gene.
  • the uraA gene codes for UraA, a membrane-bound transporter that facilitates the uptake and subsequent metabolism of the pyrimidine uracil (Andersen et al., 1995).
  • a uraA auxotroph can grow only when sufficient amounts of uracil are present, e.g., by adding uracil to growth media in vitro. Without sufficient amounts of uracil, the uraA auxotroph dies. In some embodiments, auxotrophic modifications are used to ensure that the bacteria do not survive in the absence of the auxotrophic gene product (e.g., outside of the gut). [0334] In complex communities, it is possible for bacteria to share DNA.
  • an auxotrophic bacterial strain may receive DNA from a non-auxotrophic strain, which repairs the genomic deletion and permanently rescues the auxotroph. Therefore, engineering a bacterial strain with more than one auxotroph may greatly decrease the probability that DNA transfer will occur enough times to rescue the auxotrophy.
  • the genetically engineered bacteria comprise a deletion or mutation in two or more genes required for cell survival and/or growth.
  • essential genes include, but are not limited to yhbV, yagG, hemB, secD, secF, ribD, ribE, thiL, dxs, ispA, dnaX, adk, hemH, lpxH, cysS, fold, rplT, infC, thrS, nadE, gapA, yeaZ, aspS, argS, pgsA, yefM, metG, folE, yejM, gyrA, nrdA, nrdB, folC, accD, fabB, gltX, ligA, zipA, dapE, dapA, der, hisS, ispG, suhB, tadA, acpS, era, rnc, ftsB, eno, pyrG, chpR,
  • the genetically engineered bacterium of the present disclosure is a synthetic ligand-dependent essential gene (SLiDE) bacterial cell.
  • SLiDE bacterial cells are synthetic auxotrophs with a mutation in one or more essential genes that only grow in the presence of a particular ligand (see Lopez and Anderson “Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21 (DE3 Biosafety Strain, ”ACS Synthetic Biology (2015) DOI: 10.1021/acssynbio.5b00085, the entire contents of which are expressly incorporated herein by reference).
  • the SLiDE bacterial cell comprises a mutation in an essential gene.
  • the essential gene is selected from the group consisting of pheS, dnaN, tyrS, metG and adk.
  • the essential gene is dnaN comprising one or more of the following mutations: H191N, R240C, I317S, F319V, L340T, V347I, and S345C.
  • the essential gene is dnaN comprising the mutations H191N, R240C, I317S, F319V, L340T, V347I, and S345C.
  • the essential gene is pheS comprising one or more of the following mutations: F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is pheS comprising the mutations F125G, P183T, P184A, R186A, and I188L. In some embodiments, the essential gene is tyrS comprising one or more of the following mutations: L36V, C38A and F40G. In some embodiments, the essential gene is tyrS comprising the mutations L36V, C38A and F40G. In some embodiments, the essential gene is metG comprising one or more of the following mutations: E45Q, N47R, I49G, and A51C.
  • the essential gene is metG comprising the mutations E45Q, N47R, I49G, and A51C. In some embodiments, the essential gene is adk comprising one or more of the following mutations: I4L, L5I and L6G. In some embodiments, the essential gene is adk comprising the mutations I4L, L5I and L6G. [0338] In some embodiments, the genetically engineered bacterium is complemented by a ligand.
  • the ligand is selected from the group consisting of benzothiazole, indole, 2- aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid, and L-histidine methyl ester.
  • bacterial cells comprising mutations in metG are complemented by benzothiazole, indole, 2-aminobenzothiazole, indole-3-butyric acid, indole-3-acetic acid or L-histidine methyl ester.
  • Bacterial cells comprising mutations in dnaN are complemented by benzothiazole, indole or 2- aminobenzothiazole.
  • Bacterial cells comprising mutations in pheS are complemented by benzothiazole or 2-aminobenzothiazole.
  • Bacterial cells comprising mutations in tyrS are complemented by benzothiazole or 2- aminobenzothiazole.
  • Bacterial cells comprising mutations in adk are complemented by benzothiazole or indole.
  • the genetically engineered bacterium comprises more than one mutant essential gene that renders it auxotrophic to a ligand.
  • the bacterial cell comprises mutations in two essential genes.
  • the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G) and metG (E45Q, N47R, I49G, and A51C).
  • the bacterial cell comprises mutations in three essential genes.
  • the bacterial cell comprises mutations in tyrS (L36V, C38A, and F40G), metG (E45Q, N47R, I49G, and A51C), and pheS (F125G, P183T, P184A, R186A, and I188L).
  • the genetically engineered bacterium is a conditional auxotroph whose essential gene(s) is replaced using the arabinose system described herein.
  • the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill-switch components and systems described herein.
  • the recombinant bacteria may comprise a deletion or mutation in an essential gene required for cell survival and/or growth, for example, in a DNA synthesis gene, for example, thyA, cell wall synthesis gene, for example, dapA and/or an amino acid gene, for example, serA or MetA and may also comprise a toxin gene that is regulated by one or more transcriptional activators that are expressed in response to an environmental condition(s) and/or signal(s) (such as the described arabinose system) or regulated by one or more recombinases that are expressed upon sensing an exogenous environmental condition(s) and/or signal(s) (such as the recombinase systems described herein).
  • a DNA synthesis gene for example, thyA
  • cell wall synthesis gene for example, dapA
  • an amino acid gene for example, serA or MetA
  • toxin gene that is regulated by one or more transcriptional activators that are expressed in response to an environmental condition(s) and/or signal(s
  • the genetically engineered bacterium of the disclosure is an auxotroph and also comprises kill-switch circuitry, such as any of the kill-switch components and systems described herein, as well as another biosecurity system, such a conditional origin of replication (see Wright et al., supra).
  • the disclosure provides a genetically engineered bacterium comprising an antibiotic resistance cassette.
  • the antibiotic resistance cassette is located on a plasmid in the bacterial cell. In some embodiments, the antibiotic resistance cassette is located on a chromosome in the bacterial cell. [0342] In other embodiments, the genetically engineered bacterium is cured of antibiotic resistance. In some embodiments, the genetically engineered bacterium does not comprise an exogenous antibiotic resistance cassette. In some embodiments, the recombinant bacterium does not comprise a gene encoding for antibiotic resistance. Isolated Polynucleotides [0343] In other embodiments, the disclosure provides an isolated polynucleotide encoding a uric acid catabolism enzyme. In one embodiment, the isolated polynucleotide is an isolated plasmid.
  • the disclosure provides an isolated plasmid comprising a first nucleic acid encoding a uric acid catabolism enzyme. In another embodiment, the disclosure provides an isolated plasmid comprising a second nucleic acid encoding at least one uric acid transporter. In one embodiment, the first nucleic acid and the second nucleic acid are operably linked to a first promoter. In another embodiment, the second nucleic acid is operably linked to a second promoter. [0344] In other embodiments, the disclosure provides an isolated polynucleotide or gene sequence comprising a cassette encoding a uric acid catabolism enzyme and a uric acid transporter.
  • the cassette comprises a promoter operably linked to uric acid catabolism enzyme and the uric acid transporter.
  • the cassette comprises a first and a second promoter, wherein the first promoter is operably linked to uric acid catabolism enzyme and the second promoter is operably linked to the uric acid transporter.
  • the first and/or second promoters are inducible promoters.
  • the first and second promoters are different copies of the same inducible promoter.
  • the first promoter or the second promoter, or the first promoter and the second promoter are each directly or indirectly induced by low-oxygen or anaerobic conditions.
  • first promoter or the second promoter, or the first promoter and the second promoter are each a fumarate and nitrate reduction regulator (FNR) responsive promoter.
  • FNR fumarate and nitrate reduction regulator
  • the first promoter or the second promoter, or the first promoter and the second promoter are each directly or indirectly induced by a chemical inducer.
  • the chemical inducer is IPTG.
  • the first promoter or the second promoter, or the first promoter and the second promoter are each directly or indirectly induced by temperature.
  • the inducing temperature is between 37C and 42C.
  • the isolated polynucleotide further encodes a temperature sensitive form of the CI repressor.
  • the heterologous gene encoding the uric acid catabolism enzyme and/or uric acid transporter is operably linked to a constitutive promoter.
  • a heterologous gene cassette encoding a uric acid catabolism enzyme and uric acid transporter comprises a first and a second promoter, wherein the first promoter is operably linked to the uric acid catabolism enzyme and the second promoter is operably linked to the uric acid transporter, and wherein the first and/or second promoter are constitutive promoters.
  • the constitutive promoter is a constitutive Escherichia coli ⁇ 32 promoter.
  • the constitutive promoter is a constitutive Escherichia coli ⁇ 70 promoter. In another embodiment, the constitutive promoter is a constitutive Bacillus subtilis ⁇ A promoter. In another embodiment, the constitutive promoter is a constitutive Bacillus subtilis ⁇ B promoter. In another embodiment, the constitutive promoter is a Salmonella promoter. In another embodiment, the constitutive promoter is a bacteriophage T7 promoter. In another embodiment, the constitutive promoter is and a bacteriophage SP6 promoter.
  • the plasmid further comprises a heterologous gene encoding a transporter of uric acid and/or a kill switch construct, either or both of which may be operably linked to a constitutive promoter or an inducible promoter.
  • the polynucleotide or gene sequence encoding a uric acid catabolism enzyme or a uric acid transporter is operably linked to a thermoregulated promoter, e.g., a promoter regulated by a temperature sensitive CI repressor.
  • the polynucleotide or gene sequences comprise a gene cassette encoding a uric acid catabolism enzyme and a uric acid transporter, wherein the uric acid catabolism enzyme and the uric acid transporter are each operably linked to separate copies of the same thermoregulated promoter.
  • the polynucleotide or gene sequence further encodes a temperature sensitive form of the CI repressor.
  • the polynucleotide or gene sequence encodes uric acid catabolism enzyme from C. californicus.
  • the polynucleotide or gene sequence encodes uric acid transporter uacT from E. tarda.
  • a bacterium of the disclosure may comprise a polynucleotide or gene sequence encoding a uric acid catabolism enzyme and/or a uric acid transporter operably linked to a thermoregulated promoter, e.g., a promoter regulated by a temperature sensitive CI repressor.
  • the bacterium may comprise a polynucleotide or gene sequence comprising a gene cassette encoding a uric acid catabolism enzyme and a uric acid transporter, wherein the uric acid catabolism enzyme and the uric acid transporter are each operably linked to separate copies of a thermoregulated promoter.
  • the polynucleotide or gene sequence further encodes a temperature sensitive form of the CI repressor.
  • the bacterium comprises a polynucleotide or gene sequence encoding uric acid catabolism enzyme from C. californicus.
  • the bacterium comprises a polynucleotide or gene sequence encoding uric acid transporter uacT from E. tarda.
  • the plasmid may be a high-copy plasmid. In another embodiment, the plasmid is a low-copy plasmid.
  • the disclosure provides a recombinant host cell, such as a recombinant bacterial cell, comprising an isolated plasmid described herein. In another embodiment, the disclosure provides a pharmaceutical composition comprising the recombinant bacterial cell.
  • any of the gene(s) or gene cassette(s) of the present disclosure may be integrated into the host cell chromosome, such as a bacterial chromosome, at one or more integration sites.
  • One or more copies of the gene for example, a gene encoding a uric acid catabolism enzyme or a gene encoding a uric acid transporter
  • gene cassette for example, a gene cassette comprising a gene encoding uric acid catabolism enzyme and a uric acid transporter gene
  • the host cell chromosome such as a bacterial chromosome
  • the gene encoding the uric acid catabolism enzyme and/or the uric acid transporter may be operably linked to an inducible promoter, e.g., an oxygen level-dependent or temperature sensitive promoter or an IPTG inducible promoter, may be integrated into a host cell chromosome, such as a bacterial chromosome,.
  • an inducible promoter e.g., an oxygen level-dependent or temperature sensitive promoter or an IPTG inducible promoter
  • gene encoding a uric acid catabolism enzyme and a gene encoding a uric acid transporter are arranged in a cassette, which is integrated into a host cell chromosome, such as a bacterial chromosome.
  • the gene encoding the uric acid catabolism enzyme and uric acid transporter gene are operably linked to the same copy of the same promoter.
  • the gene encoding the uric acid catabolism enzyme and uric acid transporter gene are operably linked to different promoters.
  • the gene encoding the uric acid catabolism enzyme and uric acid transporter gene are operably linked to different copies of the same promoter.
  • one or more heterologous copies of a transcriptional regulator may be integrated into the host cell chromosome, such as a bacterial chromosome.
  • the transcriptional regulator may be integrated at a separate site or as part of a gene cassette with the uric acid catabolism enzyme and/or the uric acid transporter.
  • one or more gene cassettes integrated into the host cell chromosome such as a bacterial chromosome, encoding a uric acid catabolism enzyme and/or a uric acid transporter, further comprise a gene encoding a transcriptional regulator, e.g., a LacI or a CI38 or a mutant thereof.
  • the transcriptional regulator is divergently transcribed from the same promoter region as the gene encoding the uric acid catabolism enzyme or the uric acid transporter.
  • a host cell such as a bacterium
  • the genetically engineered host cell such as a genetically engineered bacterium, comprises multiple integrated copies of the gene sequence encoding the uric acid catabolism enzyme or transporter, and/or multiple integrated copies of a gene sequence comprising a gene cassette encoding a uric acid catabolism enzyme and a uric acid transporter.
  • Multiple integrated copies may be present at the same genomic integration site, e.g., arranged in tandem or may be integrated at multiple distinct genomic integration sites.
  • multiple integrated copies of gene sequences encoding a uricase or transporter, or a gene cassette encoding a uric acid catabolism enzyme and a uric acid transporter, e.g., integrated at distinct chromosomal integration sites each are operably linked to multiple copies of the same promoters.
  • promoters are different between multiple integrated copies of the gene sequences or gene cassettes.
  • the promoters are inducible promoters, such as a low oxygen inducible FNR promoter, a temperature sensitive promoter, or an IPTG inducible promoter.
  • a bacterium of the disclosure comprises three copies of a gene cassette encoding a uric acid catabolism enzyme and a uric acid transporter. In some embodiments, each of the three copies for the gene sequences are integrated at three distinct integration sites. In some embodiments, within each of the three cassettes, the gene encoding the uric acid catabolism enzyme and the uric acid transporter gene are operably linked to different copies of the same promoter, e.g., a temperature sensitive promoter, or an IPTG inducible promoter.
  • one or more of the three gene cassettes further comprise a gene encoding a transcriptional regulator, e.g., LacI or CI38 or mutants thereof.
  • the transcriptional regulator is divergently transcribed from the same promoter region as the gene encoding the uric acid catabolism enzyme.
  • the gene sequence encodes uric acid catabolism enzyme derived from C. californicus.
  • the gene sequence encodes a uric acid transporter uacT derived from E. tarda.
  • the host cell such as a bacterium, comprises a gene sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, comprising, or consisting of SEQ ID NO: 1110.
  • the host cell such as a bacterium, comprises a gene sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, comprising, or consisting of SEQ ID NO: 1117.
  • the host cell such as a bacterium, comprises a gene sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, comprising, or consisting of SEQ ID NO: 1120.
  • the host cell such as a bacterium, comprises a gene sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, comprising, or consisting of SEQ ID NO: 1121.
  • the host cell such as a bacterium, may further comprise one or more of (1) a mutation or deletion in an endogenous prophage genome; (2) mutation or deletion in the colibactin pks island; and (3) an auxotrophic modifications, as disclosed herein.
  • the recombinant host cell such as a bacterium, are capable of consuming at least about 50 uM, 100 uM, 150 uM, 200 uM, 250 uM, 300 uM, 350 uM, 400 uM, 450 uM, 500 uM, 550 uM, 600 uM, 650 uM, 700 uM, 750 uM, 800 uM, 850 uM, 900 uM, 950 uM, or 1000 uM at 9e11 cells over 2 hours under inducing conditions, e.g., at 37 C.
  • the recombinant host cell such as a bacterium
  • a bacterium may be evaluated in vivo, e.g., in an animal model. Any suitable animal model of a disease or condition associated with uric acid, such as hyperuricemia, may be used.
  • a pig hyperuricemic gout model is described in Szczurek et al. (PLoS One, 2017, 12(6): e0179195). Young pigs, which naturally express hepatic uricase, are given nephrectomy surgery and given uric acid infusion via the juglar vein over a 7.5 hour period.
  • compositions described herein can then be given an oral dose of the pharmaceutical compositions described herein with food and water to test uric acid levels and degradation.
  • Pharmaceutical Compositions and Formulations [0366] Pharmaceutical compositions comprising the genetically engineered host cell, such as a bacterium, described herein may be used to treat, manage, ameliorate, and/or prevent a disorder associated with uric acid, e.g., hyperuricemia. Pharmaceutical compositions comprising one or more genetically engineered bacteria, alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers are provided.
  • compositions comprising the genetically engineered host cells, such as genetically engineered bacteria, of the invention may be used to treat, manage, ameliorate, and/or prevent a disorder associated with uric acid catabolism or symptom(s) associated with diseases or disorders associated with uric acid catabolism.
  • Pharmaceutical compositions of the invention comprising one or more genetically engineered host cells, such as genetically engineered bacteria, and/or one or more genetically engineered virus, alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers are provided.
  • the pharmaceutical composition comprises one species, strain, or subtype of bacteria that are engineered to comprise the genetic modifications described herein, e.g., to express a uric acid catabolism enzyme.
  • the pharmaceutical composition comprises two or more species, strains, and/or subtypes of host cell, such as a bacterium, that are each engineered to comprise the genetic modifications described herein, e.g., to express a uric acid catabolism enzyme.
  • the pharmaceutical compositions of the invention described herein may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA).
  • the pharmaceutical compositions are subjected to tabletting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. Appropriate formulation depends on the route of administration.
  • the genetically engineered host cells such as genetically engineered bacteria, may be formulated into pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g., oral, topical, injectable, intravenous, sub-cutaneous, immediate-release, pulsatile-release, delayed-release, or sustained release). Suitable dosage amounts for the genetically engineered bacteria may range from about 10 4 to 10 12 bacteria.
  • the composition may be administered once or more daily, weekly, or monthly.
  • the composition may be administered before, during, or following a meal.
  • the pharmaceutical composition is administered before the subject eats a meal. In one embodiment, the pharmaceutical composition is administered currently with a meal. In on embodiment, the pharmaceutical composition is administered after the subject eats a meal [0371]
  • the cells of the disclosure may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, buffering agents, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents.
  • the pharmaceutical composition may include, but is not limited to, the addition of calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20.
  • the genetically engineered bacteria of the invention may be formulated in a solution of sodium bicarbonate, e.g., 1 molar solution of sodium bicarbonate (to buffer an acidic cellular environment, such as the stomach, for example).
  • the genetically engineered bacteria may be administered and formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • the cells of the disclosure may be administered intravenously, e.g., by infusion or injection.
  • the cells of the disclosure may be administered intrathecally. In some embodiments, the cells of the invention may be administered orally.
  • the cells disclosed herein may be administered topically and formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well known to one of skill in the art. See, e.g., "Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA.
  • viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are employed.
  • Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, e.g., osmotic pressure.
  • auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers, or salts
  • suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle.
  • a pressurized volatile e.g., a gaseous propellant, such as freon
  • the pharmaceutical composition comprising the recombinant bacteria of the invention may be formulated as a hygiene product.
  • the hygiene product may be an antibacterial formulation, or a fermentation product such as a fermentation broth.
  • Hygiene products may be, for example, shampoos, conditioners, creams, pastes, lotions, and lip balms.
  • the cells disclosed herein may be administered orally and formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc.
  • Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose compositions such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG).
  • PVP polyvinylpyrrolidone
  • PEG polyethylene glycol
  • Disintegrating agents may also be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.
  • Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose, carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and tragacanth); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., calcium, aluminum, zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, L-leucine, magnesium stearate, talc, or silica); disintegrants (e.g., starch, potato starch, sodium starch glycolate,
  • the tablets may be coated by methods well known in the art.
  • a coating shell may be present, and common membranes include, but are not limited to, polylactide, polyglycolic acid, polyanhydride, other biodegradable polymers, alginate- polylysine-alginate (APA), alginate-polymethylene-co-guanidine-alginate (A-PMCG-A), hydroymethylacrylate-methyl methacrylate (HEMA-MMA), multilayered HEMA-MMA-MAA, polyacrylonitrilevinylchloride (PAN-PVC), acrylonitrile/sodium methallylsulfonate (AN-69), polyethylene glycol/poly pentamethylcyclopentasiloxane / polydimethylsiloxane (PEG/PD5/PDMS), poly N,N- dimethyl acrylamide (PDMAAm), siliceous encapsulates, cellulose sulphate/sodium alginate/polymethylene-co-
  • the cells disclosed herein are enterically coated for release into the gut or a particular region of the gut, for example, the large intestine.
  • the typical pH profile from the stomach to the colon is about 1-4 (stomach), 5.5-6 (duodenum), 7.3-8.0 (ileum), and 5.5-6.5 (colon).
  • the pH profile may be modified.
  • the coating is degraded in specific pH environments in order to specify the site of release. In some embodiments, at least two coatings are used. In some embodiments, the outside coating and the inside coating are degraded at different pH levels.
  • Liquid preparations for oral administration may take the form of solutions, syrups, suspensions, or a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable agents such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable
  • the preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of the genetically engineered microorganisms described herein.
  • the cells e.g., recombinant host cells, such as recombinant bacteria, of the disclosure may be formulated in a composition suitable for administration to pediatric subjects.
  • children differ from adults in many aspects, including different rates of gastric emptying, pH, gastrointestinal permeability, etc. (Ivanovska et al., Pediatrics, 134(2):361-372, 2014).
  • the composition suitable for administration to pediatric subjects may include easy-to- swallow or dissolvable dosage forms, or more palatable compositions, such as compositions with added flavors, sweeteners, or taste blockers.
  • a composition suitable for administration to pediatric subjects may also be suitable for administration to adults.
  • the composition suitable for administration to pediatric subjects may include a solution, syrup, suspension, elixir, powder for reconstitution as suspension or solution, dispersible/effervescent tablet, chewable tablet, gummy candy, lollipop, freezer pop, troche, chewing gum, oral thin strip, orally disintegrating tablet, sachet, soft gelatin capsule, sprinkle oral powder, or granules.
  • the composition is a gummy candy, which is made from a gelatin base, giving the candy elasticity, desired chewy consistency, and longer shelf-life.
  • the gummy candy may also comprise sweeteners or flavors.
  • the composition suitable for administration to pediatric subjects may include a flavor.
  • flavor is a substance (liquid or solid) that provides a distinct taste and aroma to the formulation. Flavors also help to improve the palatability of the formulation. Flavors include, but are not limited to, strawberry, vanilla, lemon, grape, bubble gum, and cherry.
  • the genetically engineered microorganisms may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject’s diet.
  • the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • a compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
  • the pharmaceutical composition comprising the cells disclosed herein may be a comestible product, for example, a food product.
  • the food product is milk, concentrated milk, fermented milk (yogurt, sour milk, frozen yogurt, lactic acid bacteria- fermented beverages), milk powder, ice cream, cream cheeses, dry cheeses, soybean milk, fermented soybean milk, vegetable-fruit juices, fruit juices, sports drinks, confectionery, candies, infant foods (such as infant cakes), nutritional food products, animal feeds, or dietary supplements.
  • the food product is a fermented food, such as a fermented dairy product.
  • the fermented dairy product is yogurt.
  • the fermented dairy product is cheese, milk, cream, ice cream, milk shake, or kefir.
  • the recombinant bacteria of the invention are combined in a preparation containing other live bacterial cells intended to serve as probiotics.
  • the food product is a beverage.
  • the beverage is a fruit juice-based beverage or a beverage containing plant or herbal extracts.
  • the food product is a jelly or a pudding.
  • Other food products suitable for administration of the recombinant bacteria of the invention are well known in the art. For example, see U.S.2015/0359894 and US 2015/0238545, the entire contents of each of which are expressly incorporated herein by reference.
  • the pharmaceutical composition of the invention is injected into, sprayed onto, or sprinkled onto a food product, such as bread, yogurt, or cheese.
  • the composition is formulated for intraintestinal administration, intrajejunal administration, intraduodenal administration, intraileal administration, gastric shunt administration, or intracolic administration, via nanoparticles, nanocapsules, microcapsules, or microtablets, which are enterically coated or uncoated.
  • the pharmaceutical compositions may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • the compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and/or dispersing agents.
  • the cells e.g., recombinant bacterial cells, described herein may be administered intranasally, formulated in an aerosol form, spray, mist, or in the form of drops, and conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • Pressurized aerosol dosage units may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the cells e.g., recombinant bacterial cells, may be administered and formulated as depot preparations. Such long acting formulations may be administered by implantation or by injection, including intravenous injection, subcutaneous injection, local injection, direct injection, or infusion.
  • compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).
  • suitable polymeric or hydrophobic materials e.g., as an emulsion in an acceptable oil
  • ion exchange resins e.g., as an emulsion in an acceptable oil
  • sparingly soluble derivatives e.g., as a sparingly soluble salt.
  • a single dosage form may be administered in bolus form, e.g., single injection, single oral dose, including an oral dose that comprises multiple tablets, capsule, pills, etc.
  • a single dosage form may be administered over a period of time, e.g., by infusion.
  • Single dosage forms of the pharmaceutical composition may be prepared by portioning the pharmaceutical composition into smaller aliquots, single dose containers, single dose liquid forms, or single dose solid forms, such as tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated.
  • a single dose in a solid form may be reconstituted by adding liquid, typically sterile water or saline solution, prior to administration to a patient.
  • the composition can be delivered in a controlled release or sustained release system.
  • a pump may be used to achieve controlled or sustained release.
  • polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see e.g., U.S. Patent No.5,989,463).
  • polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N- vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters.
  • the polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable.
  • a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used. [0389] Dosage regimens may be adjusted to provide a therapeutic response. Dosing can depend on several factors, including severity and responsiveness of the disease, route of administration, time course of treatment (days to months to years), and time to amelioration of the disease.
  • a single bolus may be administered at one time, several divided doses may be administered over a predetermined period of time, or the dose may be reduced or increased as indicated by the therapeutic situation.
  • the specification for the dosage is dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved. Dosage values may vary with the type and severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the treating clinician. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or animal models.
  • LD50, ED50, EC50, and IC50 may be determined, and the dose ratio between toxic and therapeutic effects (LD50/ED50) may be calculated as the therapeutic index.
  • Compositions that exhibit toxic side effects may be used, with careful modifications to minimize potential damage to reduce side effects.
  • Dosing may be estimated initially from cell culture assays and animal models. The data obtained from in vitro and in vivo assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the pharmaceutical compositions may be packaged in a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent.
  • one or more of the pharmaceutical compositions is supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject.
  • one or more of the prophylactic or therapeutic agents or pharmaceutical compositions is supplied as a dry sterile lyophilized powder in a hermetically sealed container stored between 2° C and 8° C and administered within 1 hour, within 3 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, or within one week after being reconstituted.
  • Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%).
  • Other suitable cryoprotectants include trehalose and lactose.
  • Suitable bulking agents include glycine and arginine, either of which can be included at a concentration of 0-0.05%, and polysorbate-80 (optimally included at a concentration of 0.005-0.01%).
  • Additional surfactants include but are not limited to polysorbate 20 and BRIJ surfactants.
  • the pharmaceutical composition may be prepared as an injectable solution and can further comprise an agent useful as an adjuvant, such as those used to increase absorption or dispersion, e.g., hyaluronidase.
  • the genetically engineered viruses are prepared for delivery, taking into consideration the need for efficient delivery and for overcoming the host antiviral immune response.
  • Approaches to evade antiviral response include the administration of different viral serotypes as part of the treatment regimen (serotype switching), formulation, such as polymer coating to mask the virus from antibody recognition and the use of cells as delivery vehicles.
  • the composition can be delivered in a controlled release or sustained release system.
  • a pump may be used to achieve controlled or sustained release.
  • polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see e.g., U.S. Patent No.5,989,463).
  • polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N- vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters.
  • the polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable.
  • a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used.
  • the cells e.g., recombinant bacterial cells, may be administered and formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • Methods of Treatment [0395] Further disclosed herein are methods of treating diseases associated with uric acid. In some embodiments, disclosed herein are methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these diseases or disorders.
  • disease associated with uric acid or “disorder associated with uric acid catabolism” is a disease or disorder involving the abnormal, e.g., increased, levels of uric acid in a subject.
  • a disease or disorder associated with uric acid is hyperuricemia.
  • a disease or disorder associated with uric acid is gout.
  • the disclosure provides methods for reducing, ameliorating, or eliminating one or more symptom(s) associated with these diseases.
  • Gout is a form of inflammatory arthritis characterized by sudden, severe attacks of pain and swelling in the joints, caused by high serum levels of uric acid.
  • Gout affects 8.3 million people in the United States, alone, and its prevalence is estimated at 3.9% of the population. Gout is also a prognostic indicator of renal disease, type I diabetes, and cardiovascular disease.
  • gout is treated with dietary intervention and drugs that inhibit purine synthesis (such as Allopurinol) and promote renal excretion (such as Benzbromarone), and it is estimated that 2 million people in the United States, alone, take medication to decrease serum uric acid levels.
  • purine synthesis such as Allopurinol
  • Benzbromarone renal excretion
  • the method may comprise preparing a pharmaceutical composition with at least one genetically engineered species, strain, or subtype of bacteria described herein, and administering the pharmaceutical composition to a subject in a therapeutically effective amount.
  • the genetically engineered bacteria disclosed herein are administered orally, e.g., in a liquid suspension.
  • the genetically engineered bacteria are lyophilized in a gel cap and administered orally.
  • the genetically engineered bacteria are administered via a feeding tube or gastric shunt.
  • the genetically engineered bacteria are administered rectally, e.g., by enema.
  • the genetically engineered bacteria are administered topically, intraintestinally, intrajejunally, intraduodenally, intraileally, and/or intracolically. In one embodiment, the genetically engineered bacteria are injected directly into a tumor. [0399] In certain embodiments, administering the pharmaceutical composition to the subject reduces the level of uric acid in a subject. In some embodiments, the methods of the present disclosure may reduce the level of uric acid in a subject by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to levels in an untreated or control subject.
  • reduction is measured by comparing the uric acid concentration in a subject before and after administration of the pharmaceutical composition.
  • the method of treating or ameliorating a disease or disorder allows one or more symptoms of the condition or disorder to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.
  • Uric acid levels may be measured by methods known in the art (see uric acid catabolism enzyme section, supra).
  • uric acid concentrations in the subject may be measured in a biological sample, such as blood, serum, plasma, urine, fecal matter, peritoneal fluid, intestinal mucosal scrapings, a sample collected from a tissue, and/or a sample collected from the contents of one or more of the following: the stomach, duodenum, jejunum, ileum, cecum, colon, rectum, and anal canal.
  • a biological sample such as blood, serum, plasma, urine, fecal matter, peritoneal fluid, intestinal mucosal scrapings, a sample collected from a tissue, and/or a sample collected from the contents of one or more of the following: the stomach, duodenum, jejunum, ileum, cecum, colon, rectum, and anal canal.
  • the methods may include administration of the compositions to reduce uric acid concentrations in a subject to undetectable levels, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% of the subject’s uric acid concentration(s) prior to treatment.
  • the methods disclosed herein may further comprise isolating a sample from the subject prior to administration of a composition and determining the level of the uric acid in the sample.
  • the methods may further comprise isolating a sample from the subject after administration of a composition and determining the level of uric acid in the sample.
  • a recombinant bacterium disclosed herein is capable of producing allantoin, which can be used as a biomarker to determine treatment efficacy.
  • the genetically engineered host cells such as genetically engineered bacteria, comprising a uric acid catabolism enzyme is E. coli Nissle.
  • the genetically engineered bacteria may be destroyed, e.g., by defense factors in the gut or blood serum (Sonnenborn et al., 2009), or by activation of a kill switch, several hours or days after administration.
  • the pharmaceutical composition may be re-administered at a therapeutically effective dose and frequency. Length of Nissle residence in vivo in mice can be determined.
  • the genetically engineered bacteria are not destroyed within hours or days after administration and may propagate and colonize the gut.
  • the methods disclosed herein may comprise administration of a composition alone or in combination with one or more additional therapies.
  • the pharmaceutical composition may be administered alone or in combination with one or more additional therapeutic agents.
  • the methods may also comprise following a restricted diet.
  • Urate abundance from natural sources of protein ranges from 30% accessibility within the gastrointestinal track (210 -600mg). Assuming the average human subject needs to degrade about 600mg of urate per day with meals, and assuming the recombinant bacteria provides 4 hours of activity per dose, that leaves 3x doses per day at 5x10 11 dose and 600mg urate per day (200mg/dose).
  • 200mg urate /dose 1200 ⁇ mol urate. 1200 ⁇ mol/4 hours/5x10 11 cells leads to 0.6 ⁇ mol/hr/1x10 9 cells.
  • the target dose is 5x10 11 live recombinant bacterial cells / mL.
  • the recombinant bacteria disclosed herein has a urate degradation activity of about 0.05 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 0.06 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 0.07 ⁇ mol/hr/1x10 9 cells.
  • the recombinant bacteria disclosed herein has a urate degradation activity of about 0.08 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 0.09 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 0.1 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 0.15 ⁇ mol/hr/1x10 9 cells.
  • the recombinant bacteria disclosed herein has a urate degradation activity of about 0.2 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 0.25 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 0.3 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 0.4 ⁇ mol/hr/1x10 9 cells.
  • the recombinant bacteria disclosed herein has a urate degradation activity of about 0.5 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 0.6 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 0.7 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 0.8 ⁇ mol/hr/1x10 9 cells.
  • the recombinant bacteria disclosed herein has a urate degradation activity of about 0.9 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 1.0 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 1.10 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 1.30 ⁇ mol/hr/1x10 9 cells.
  • the recombinant bacteria disclosed herein has a urate degradation activity of about 1.35 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 1.40 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 1.45 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 1.50 ⁇ mol/hr/1x10 9 cells.
  • the recombinant bacteria disclosed herein has a urate degradation activity of about 0.06 ⁇ mol/hr/1x10 9 cells to about .9 umol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 0.12 ⁇ mol/hr/1x10 9 cells to about 0.9 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 0.06 ⁇ mol/hr/1x10 9 cells to about 0.84 umol/hr/1x10 9 cells.
  • the recombinant bacteria disclosed herein has a urate degradation activity of about 0.24 ⁇ mol/hr/1x10 9 cells to about 0.66 umol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 0.06 ⁇ mol/hr/1x10 9 cells to about 0.60 ⁇ mol/hr/1x10 9 cells. Accordingly, in one embodiment, the recombinant bacteria disclosed herein has a urate degradation activity of about 0.3 ⁇ mol/hr/1x10 9 cells to about 0.90 ⁇ mol/hr/1x10 9 cells.
  • the recombinant bacteria disclosed herein has a urate degradation activity of about 0.5 ⁇ mol/hr/1x10 9 cells to about 0.75 ⁇ mol/hr/1x10 9 cells.
  • about 60 mg to about 600 mg of urate are degraded per day.
  • about 6 mg to about 900 mg of urate are degraded per day.
  • about 60mg of urate are degraded per day.
  • about 120 mg of urate are degraded per day.
  • about 180 mg of urate are degraded per day.
  • about 240 mg of urate are degraded per day.
  • about 300 mg of urate are degraded per day. In one embodiment, about 360 mg of urate are degraded per day. In one embodiment, about 420 mg of urate are degraded per day. In one embodiment, about 480 mg of urate are degraded per day. In one embodiment, about 540 mg of urate are degraded per day. In one embodiment, about 600 mg of urate are degraded per day. In one embodiment, about 660 mg of urate are degraded per day. In one embodiment, about 720 mg of urate are degraded per day. In one embodiment, about 780 mg of urate are degraded per day. In one embodiment, about 840 mg of urate are degraded per day.
  • the agent(s) should be compatible with the genetically engineered bacteria disclosed herein, e.g., the agent(s) must not kill the bacteria.
  • the pharmaceutical composition is administered with food. In alternate embodiments, the pharmaceutical composition is administered before or after eating food.
  • the pharmaceutical composition may be administered in combination with one or more dietary modifications, e.g., low-protein diet.
  • the dosage of the pharmaceutical composition and the frequency of administration may be selected based on the severity of the symptoms and the progression of the disorder. The appropriate therapeutically effective dose and/or frequency of administration can be selected by a treating clinician.
  • Example 1 Strain Development and Testing
  • Prototype strains included two plasmids: 1) a low-copy pSC101 plasmid containing a urate transporter – uacT – from E.
  • Plasmids were constructed through TypeIIS cloning of synthesized gBlock fragments (IDT, Coralville, IA) containing these genes, followed by Sanger sequencing for sequence verification.
  • E. coli Nissle E. coli Nissle
  • EcN strains harboring the urate transporter and uric acid degrading-enzyme plasmids were grown to early log phase and induced for expression with 200ng/mL ATC. Induction is allowed to proceed for 4h, at which time cells are harvested by centrifugation and biomass stored in PBS containing 15% glycerol at -80 o C.
  • Example 2 Strain Activity Calculation [0413] Urate abundance in natural sources of protein ranges from 30% accessibility within the gastrointestinal track (210 -600mg).
  • Gout is a form of inflammatory arthritis caused by high levels of uric acid (urate, UA) in serum (hyperuricemia) and characterized by sudden, severe attacks of pain and swelling in the joints. Affecting 8.3 million people in the US, gout is a prognostic indicator of joint damage, bone loss, tophi, renal disease, type 1 diabetes, and cardiovascular disease (Dalbeth et al, Nat Rev Dis Primers, 5(1): 69 (2019)).
  • uric acid is produced in the liver. Once produced, 70% of the uric acid is excreted by the kidney while 30% enters the gastrointestinal (GI) tract.
  • GI gastrointestinal
  • HIU 2- oxo-4-hydroxy-4-carboxy-5-ureidoimidazoleoline
  • OHCU 2- oxo-4-hydroxy-4-carboxy-5-ureidoimidazoleoline
  • FIU allantoin
  • FIG. 1 Two putative oxidoreductases (aegA and ygfT) were discovered to be involved in the degradation of uric acid under anaerobic and microaerobic conditions in E. coli; however, the chemistry, molecular mechanism, and end-products are unknown (K Iwadate and J Kato, J Bacteriol, 201:11, (2019)). Under anaerobic and microaerobic conditions, E. coli can use uric acid as a sole source of nitrogen.
  • Escherichia coli Nissle (EcN) strains have been successfully modified to degrade specific dietary amino acids from within the gut in order to reduce the systemic abundance of the specific amino acids in patients with genetic inborn errors of amino acid metabolism.
  • EcN Escherichia coli Nissle
  • phenylalanine- and leucine-degrading EcN have been constructed for the treatment of phenylketonuria (PKU) and maple syrup urine disease (MSUD), respectively. These engineered EcN have shown measurable activity against their target metabolite in preclinical in vivo models (and in human patients in the case of PKU).
  • a uric acid degrading EcN could be efficacious for the treatment of gout.
  • the ideal uricase candidate may be one that functions well at lower oxygen tension rather than one with a high rate of activity in highly aerobic conditions.
  • the capacity of the overexpression of E. coli putative oxidoreductases, AegA and YgfT, to degrade uric acid in EcN via an uncharacterized microaerobic/anaerobic pathway is also investigated. This pathway can be further elucidated including other key members of the reaction and the end products.
  • the sequence of the endogenous EcN homologs implicated in E. coli uric acid degradation (K Iwadate and J Kato, J Bacteriol, 201:11, (2019)) are given in the Sequence Listing below.
  • E. coli employs a uric acid specific proton symporter, ygfU (FIGs.3A and 3B), a member of the ubiquitous nucleobase-ascorbate transporter family (NCS2). See K Papakostas and S Frillingos, J Biol Chem, 287(19): 15684-95, (2012).
  • UA degradation via uricase yields allantoin in a 1:1 stochiometric ratio (FIG.1). Biodistribution of allantoin was investigated, and any metabolites formed from allantoin might be considered as potential biomarkers of strain activity in vivo. [0427] It is important to note here that all activity of strains was measured as “Activated Biomass.” This more closely mimics the activity of the live biotherapeutic in a clinical setting. Briefly, cells were grown to early log phase and uricase is induced for 4h. Cells were subsequently spun down, harvested, and stored in glycerol at -80 o C until testing.
  • coli Nissle (FIG.4) comprising a low-copy pSC101 plasmid containing a urate transporter – uacT – from E. coli MG1655, which will increase import of urate/uric acid into the cell, and 2) a medium-copy p15a plasmid containing a uricase enzyme (from Candida utilis) was measured in comparison to the control strain, SYN094 (E. coli Nissle control), and to uricase (Candida sp.) (FIGs.5A and 5B, Tables 6 and 7). Lysate from SYN7229 (C. utilis uricase (SEQ ID NO: 228), E.
  • Activated biomass was made by growing 2 mL overnight bacterial cultures in LB media containing 5 mM uric acid (UA) under microaerobic (5-10% oxygen) conditions. The following day, cultures were back diluted 1:100 in 15 mL fresh LB media containing 5 mM UA and grown microaerobically for 2hrs at 37 ⁇ C with shaking at 250 rpm. After 2hrs of growth, cells were induced with 2X ATC (anhydrotetracycline) and grow for an additional 4hrs at 37 ⁇ C with shaking at 250 rpm. After 6hrs of total growth, bacterial cells were pelleted by centrifugation at 8000 rpm for 5mins.
  • 2X ATC anhydrotetracycline
  • a volume of cells/lysate equivalent to an OD of 1 was added to 1 mL of M9 minimal media containing 0.5% glucose and 1 mM UA in a 1.7 mL Eppendorf tube. Tubes were vortexed briefly to evenly distribute cells/lysate. Tubes were placed at 37 ⁇ C with no shaking. At 0.5, 1.0, 1.5, 2.0, and 4.0hr timepoints, 150 ⁇ L of cell/lysate and media suspension were removed and spun down at high speed for ⁇ 1min to pellet cells and 100 ⁇ L of supernatant was added to a well of flat-bottom 96-well plate compatible with plate reader. Absorbance at 290 nm was measured as UA has strong absorbance characteristics at 290 nm.
  • coli MG1655 transporter (SEQ ID NO: 10)) comprising a low-copy (e.g., 3-5 copies/cell) pSC101 plasmid containing a urate transporter (uacT) from E. coli MG1655, and a medium-copy p15a plasmid (e.g., 10-15 copies) containing a uricase enzyme (Candida utilis) was measured in comparison to the control strain, SYN094 (E. coli Nissle control), for excretion in urine in two different studies in an acute mouse model of hyperuricosuria (FIGs.6A-6D).
  • SYN094 E. coli Nissle control
  • Loop Culture grew overnight for ⁇ 14-16h in 50 mL of FM3/25g/L glucose/ carbenicillin/Kanamycin medium in a 500-mL baffled flask. The Flask was incubated at 37 o C and mixed at 350 RPM. Next day, Seed culture of ⁇ 20-40 OD600 and was used to inoculate a fermenter vessel with FM3/25g/L glucose/ carbenicillin/Kanamycin medium to a starting OD600 of 0.18. The fermentation was grown at 37 o C at pH 7 with dissolved oxygen setpoint of 60% for ⁇ 6 hours to achieve final biomass production. The fermentation growth phase was about 2h until the OD600 was ⁇ 4 OD600.
  • the culture was induced to a 2X ATC concentration to activate the cells.
  • the induction of cells continued for four hours until the generation of final biomass reached between 20-30 OD600.
  • Fermentation was harvested at their targeted OD during 4h post induction endpoint and spun down by centrifuging culture broth for 30 min at 4500 RPM at 4 o C. They were finally resuspended at a 6-7X concentration in PKU buffer, so cell concentration was above 1e11.
  • the Liquid formulation was aliquoted and stored at -80 o C.
  • coli Nissle control in a 3L fermenter (first study), a seed flask fermentation was started from a scraping of the frozen MCB culture in a cryovial with an inoculum loop and added to FM3/30g/L glycerol/ Strep media. Loop Culture grew overnight for ⁇ 14- 16h in 50 mL of FM3/30g/L glycerol / Strep medium in a 500-mL baffled flask. The Flask was incubated at 37°C and mixed at 350 RPM.
  • Seed culture of ⁇ 30-60 OD600 was used to inoculate a fermenter vessel with FM3/30g/L glycerol / Strep medium to a starting OD600 of 0.18.
  • the 1.5L in a 3L fermentation tank was grown at 37 o C at pH 7 with dissolved oxygen setpoint of 60% for ⁇ 5 hours to achieve final biomass production.
  • the fermentation growth phase was about 2h until the OD600 was ⁇ 2-4 OD600.
  • the culture was induced to a 2X ATC concentration to simulated activation conditions.
  • the addition of inducer solution was to match the candidate expression strain condition only.
  • Fermentation was harvested at their targeted OD during 4h post induction endpoint and spun down by centrifuging culture broth for 30 min at 4500 RPM at 4 o C. They were finally resuspended at a 6-7X concentration in PKU buffer, so cell concentration was above 1e11.
  • the Liquid formulation was aliquoted and stored at -80 o C.
  • SYN094 E. coli Nissle control
  • AMBR fermenter second study
  • a seed flask fermentation was started from a scraping of the frozen MCB culture in a cryovial with an inoculum loop and added to FM3/25g/L glucose/ Strep media.
  • Loop Culture grew overnight for ⁇ 14-16h in 50 mL of FM3/25g/L glucose/ Strep medium in a 500-mL baffled flask.
  • the Flask was incubated at 37 o C and mixed at 350 RPM.
  • a seed culture of ⁇ 30-60 OD600 was used to inoculate a fermenter vessel with FM3/25g/L glucose/ Strep medium to a starting OD600 of 0.18.
  • the fermentation was grown at 37 o C at pH 7 with dissolved oxygen setpoint of 60% for ⁇ 5 hours to achieve final biomass production.
  • the fermentation completely growth phase for ⁇ 5h until the OD600 was ⁇ 30-40 OD600. There was no induction stage for these cells since the strain is a control host chassis.
  • mice were kept on fasting throughout the duration of the studies and were administered orally using a flexible feeding tube attached to a sterile single use syringe with 200 ⁇ l of vehicle (glycerol/PBS) or 2 x 10 10 SYN094 (E.
  • mice were orally gavaged with 200 ⁇ L of labeled uric acid (uric acid-1,3-15N298 atom % 15N) at dose 50 mg/Kg.
  • Urine samples were collected at 2 hours following uric acid dosing. Samples were analyzed using LC-MS/MS, and concentrations of labelled uric acid measured and normalized by excreted creatinine (ug uric acid/mg Creatinine).
  • mice treated with SYN7229 C. utilis uricase (SEQ ID NO: 228), E. coli MG1655 transporter (SEQ ID NO: 10)
  • SYN094 E. coli Nissle control
  • glycerol FIG.6B
  • mice receiving the vehicle excreted a higher concentration of uric acid in the urine while mice treated with SYN7229 and SYN094 (E.
  • Uricase screening using a metagenomics library screen resulted in several potential uricase genes of interest (FIG.7). These optimized uricase enzymes were cloned and tested in several assays. [0441] In vitro assays, as described above, showed multiple uricase and transporter combinations were capable of decreasing uric acid in media.
  • FIG.8 demonstrates that strain 851796 (SYN7957) (C. californicus uricase (SEQ ID NO: 219), E. ictaluri transporter (SEQ ID NO: 220)) decreased uric acid close to 100% in one hour.
  • Strains 851714 C. utilis v1 uricase-Ptet (SEQ ID NO: 228), E. ictaluri transporter-PlacIO (SEQ ID NO: 220)
  • 851573 A. globiformis uricase (SEQ ID NO: 218)
  • Strain 851774 Mus musculus uricase (SEQ ID NO: 230), E.
  • FIG.9A demonstrates that strains with uricase (without a transporter) decreased uric acid levels in media. Strain 870791 (C. californicus uricase (SEQ ID NO: 219)) minimally decreased uric acid, while strains 776000 (C. utilis uricase-Ptet (SEQ ID NO: 228)) and 890735 (C.
  • FIG.9B demonstrates that strains with uricase (with a transporter) decreased uric acid in media.
  • strains 851796 SYN7957
  • C. californicus uricase SEQ ID NO: 219
  • E. ictaluri transporter SEQ ID NO: 220
  • 771295 SYN7229
  • C. utilis uricase SEQ ID NO: 228, E.
  • FIG.11 demonstrates uric acid consumption by E. coli strains at 37 °C with or without shaking.
  • Strains 851714 C. utilis v1 uricase-Ptet (SEQ ID NO: 228), E.
  • ictaluri transporter-PlacIO SEQ ID NO: 220
  • 851752 A. globiformis uricase (SEQ ID NO: 218)
  • 851796 SYN7957
  • C. californicus uricase SEQ ID NO: 219
  • E. ictaluri transporter (SEQ ID NO: 220)) decreased uric acid about 2-fold in 1 hours and about 4.5-fold in 2 hours when the culture was shaking.
  • strains 851714 C. utilis v1 uricase-Ptet (SEQ ID NO: 228)
  • ictaluri transporter-PlacIO SEQ ID NO: 220
  • 851752 A. globiformis uricase (SEQ ID NO: 218), E. ictaluri transporter (SEQ ID NO: 220)), and 851796 (SYN7957) (C. californicus uricase (SEQ ID NO: 219), E. ictaluri transporter (SEQ ID NO: 220)) minimally decreased uric acid.
  • SYN7229 C. utilis uricase (SEQ ID NO: 228), E. coli MG1655 transporter (SEQ ID NO: 10)
  • FIG.12 shows uric acid consumption by E. coli strains at 37 °C with or without shaking.
  • Strain SYN7897 C.
  • FIG.13 demonstrates uric acid consumption by E. coli strains at 37 °C with or without shaking.
  • Strain SYN7960 C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)
  • SYN7960 C. californicus uricase (plasmid) (SEQ ID NO: 219)
  • E. tarda transporter (plasmid) SEQ ID NO: 222)
  • californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)) decreased uric acid in the media about 1.25-fold and about 2-fold after 1 hour and 2 hours, respectively, without shaking.
  • Strains SYN7957 (C. californicus uricase (SEQ ID NO: 219), E. ictaluri transporter (SEQ ID NO: 220)) and SYN7997 (C. utilis uricase-PlacIO (SEQ ID NO: 228), E. tarda transporter-PlacIO (SEQ ID NO: 222)) decreased uric acid in the media about 1.4-fold and about 1.3-fold, respectively, with shaking.
  • FIG.14 demonstrates uric acid consumption by E. coli strains without shaking compared to a control strain (SYN094 (E.
  • tarda transporter (SEQ ID NO: 222), ⁇ appC) decreased uric acid in media about 1.6-fold and about 2.5-fold in 1 hour and two hours, respectively, when not shaking and compared to the control strain (SYN094 (E. coli Nissle control)).
  • Strains SYN7997 (C. utilis uricase-PlacIO (SEQ ID NO: 228), E. tarda transporter-PlacIO (SEQ ID NO: 222)), SYN7957 (C. californicus uricase (SEQ ID NO: 219), E. ictaluri transporter (SEQ ID NO: 220)) and SYN7959 (890902) (C.
  • uricase-PlacIO (SEQ ID NO: 228), E. ictaluri transporter-PlacIO (SEQ ID NO: 220)) decreased uric acid in the media about 1.25-fold in two hours without shaking and compared to a control strain (SYN094 (E. coli Nissle control)).
  • SYN094 E. coli Nissle control
  • Simulated intestinal fluid (SIF) assays examined the uric acid consumption activity (FIG. 15A) and allantoin production activity (FIG.15B) of several strains in conditions similar to the mammalian gut.
  • SYN7960 C. californicus uricase (plasmid) (SEQ ID NO: 219), E.
  • tarda transporter (plasmid) (SEQ ID NO: 222)), SYN8012 (C. californicus uricase (SEQ ID NO: 219), E. tarda transporter (SEQ ID NO: 222), ⁇ appB), and SYN8013 (C. californicus uricase (SEQ ID NO: 219), E. tarda transporter (SEQ ID NO: 222), ⁇ appC) produced about 0.8 mM allantoin in two hours, compared to no allantoin produced by the control strain, SYN094 (E. coli Nissle control). Strain SYN7957 (C. californicus uricase (SEQ ID NO: 219), E.
  • FIG.16 demonstrates uric acid consumption by SYN094 (E. coli Nissle control), SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)), SYN8196 (C. californicus uricase (integrated) (SEQ ID NO: 219)), and SYN8197 (C.
  • californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222)).
  • SYN8197 C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222)) and SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)) degraded uric acid at a rate of 0.3 umol/1E9 cells/hr and 0.74 umol/1E9 cells/hr, respectively.
  • tarda transporter (plasmid) (SEQ ID NO: 222) was administered to non-human primates (NHP) at a dose of 1E11 live cells (LCs) with 15N-uric acid. Animals fasted overnight and overnight urine and feces were set aside. At T0, the animals were given SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)) or SYN094 (E. coli Nissle control) (control) and 15N- UA. Blood samples were taken 1 hour prior to administration, and at 30 mins, 1 hour, 2 hours, 4 hours, and 6 hours after administration.
  • SYN7960 C. californicus uricase (plasmid) (SEQ ID NO: 219)
  • E. tarda transporter (plasmid)
  • SYN094 E. coli Nissle control
  • FIG.17A demonstrates 15N-UA from cumulative urine samples 6 hours after administration of SYN094 (E. coli Nissle control) or SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)).
  • SYN7960 C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)
  • FIG.17B demonstrates endogenous uric acid in urine samples decreased about 1.3-fold when SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)) was administered compared to the control SYN094 (E. coli Nissle control).
  • FIG.18A demonstrates SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)) decreased 15N-UA in cumulative feces samples after 6 hours by about 6-fold when compared to SYN094 (E.
  • FIG.19A demonstrates 15N-UA from cumulative urine samples 6 hours after administration of SYN094 (E. coli Nissle control) or SYN7229 (C. utilis uricase (SEQ ID NO: 228), E. coli MG1655 transporter (SEQ ID NO: 10)).
  • SYN7229 C. utilis uricase (SEQ ID NO: 228), E. coli MG1655 transporter (SEQ ID NO: 10)
  • SYN094 E. coli Nissle control
  • FIG. 19B demonstrates endogenous uric acid in urine samples decreased about 5-fold when SYN7229 (C. utilis uricase (SEQ ID NO: 228), E. coli MG1655 transporter (SEQ ID NO: 10)) was administered compared to the control SYN094 (E. coli Nissle control).
  • Example 9 Uric Acid Consumption and Allantoin Production Assays with Optimized Strains
  • SYN-GOUT SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222))
  • SYN-GOUT SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)
  • SYN-GOUT SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E.
  • californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)); SYN8197 (C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222)); SYN8278 (C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222), C. californicus uricase (plasmid) (SEQ ID NO: 219); ⁇ clb ⁇ dapA ⁇ ); SYN8279 (C.
  • californicus uricase integrated (SEQ ID NO: 219)
  • E. tarda transporter integrated (SEQ ID NO: 222)
  • E. tarda transporter plasmid
  • SEQ ID NO: 222 ⁇ clb ⁇ dapA ⁇
  • SYN094 E. coli Nissle control
  • Uric acid consumption decreases with uricase and transporter genes are only integrated on the bacterial chromosome (FIG.21).
  • SYN7960 C. californicus uricase (plasmid) (SEQ ID NO: 219)
  • tarda transporter (plasmid) (SEQ ID NO: 222)
  • uric acid is decreased by about 20% after 2 hours when compared to the SYN094 (E. coli Nissle control) control.
  • SYN8197 C. californicus uricase (integrated) (SEQ ID NO: 219)
  • E. tarda transporter (integrated) SEQ ID NO: 222)
  • SYN8278 C. californicus uricase (integrated) (SEQ ID NO: 219)
  • E. tarda transporter (integrated) (SEQ ID NO: 222), C.
  • californicus uricase (plasmid) (SEQ ID NO: 219); ⁇ clb ⁇ dapA ⁇ ), and SYN8279 (C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222), E. tarda transporter (plasmid) (SEQ ID NO: 222); ⁇ clb ⁇ dapA ⁇ ) decrease uric acid in the media between at least about 60% to at least about 70% after 2 hours when compared to the control strain.
  • Allantoin production is also increased when uricase and/or transporter genes are present on a plasmid without a second copy integrated on the bacterial chromosome when compared to strains comprising uricase and transporter genes integrated on the bacterial chromosome only (FIG.22).
  • SYN8197 C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222)
  • SYN8278 C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222), C.
  • FIG.25A compared uric acid consumption between SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)) (comprising uricase and transporter genes on a plasmid), SYN8581 (C. californicus uricase (integrated) (SEQ ID NO: 219), E.
  • FIGs.26A and 26B show that the rate of uric acid consumption is increased with increasing copies of uricase and transporter genes integrated in the bacterial chromosome.
  • EcN represents wild-type, unengineered E.
  • PLASMID corresponds to SYN7960 (C. californicus uricase (plasmid) (SEQ ID NO: 219), E. tarda transporter (plasmid) (SEQ ID NO: 222)), “1 st OPT.” corresponds to SYN8581 (C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222); 1 copy each, Pr/L promoter and cI38 repressor; ⁇ clb ⁇ dapA ⁇ ), “2 nd OPT.” corresponds to SYN8592 (C.
  • californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222); 2 copies each; ⁇ clb ⁇ dapA ⁇ ), and “3 rd OPT.” corresponds to SYN8634 (C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222); 3 copies each; ⁇ clb ⁇ dapA ⁇ ).
  • Activated biomass was made by growing 2 mL overnight bacterial cultures in LB media. The following day, cultures were back diluted 1:100 in 20 mL fresh LB media and grown for 2hrs at 30 ⁇ C with shaking at 250 rpm.
  • a volume of cells/lysate equivalent to an OD of 1 was added to 1 mL of M9 minimal media containing 1 mM UA in a 1.7 mL Eppendorf tube. Tubes were vortexed briefly to evenly distribute cells/lysate. Tubes were placed at 37 ⁇ C with no shaking. At 0.5, 1.0, 1.5, and 2.0 timepoints, 150 ⁇ L of cell/lysate and media suspension were removed and spun down at high speed for ⁇ 1min to pellet cells and 100 ⁇ L of supernatant was added to a well of flat-bottom 96-well plate compatible with plate reader. Absorbance at 290 nm was measured as UA has strong absorbance characteristics at 290 nm.
  • Results are shown in FIGs.31C and 31D. They demonstrate that IP administered 15N-UA can be recovered both in the plasma and in the small intestine when potassium oxanate was included. The recovery of 15N-UA in the small intestine indicates the existence of an enterorecirculation loop for uric acid between the systemic circulation and the gut. Very little 15N-UA was detected in plasma or small intestine in the absence of potassium oxonate, indicating the presence of a highly active endogenous uricase in mice.
  • Example 11 Assessment of Strain Activity in a Two-week Crossover study in Non-Human Primates with transient elevation of uricemia [0467] This study assessed whether SYN8669 (C.
  • californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222); 3 copies each; cured of antibiotic resistance; ⁇ clb ⁇ dapA ⁇ ) actively consumed uric acid from within the gut in non-human primates.
  • Test Article was administered to the appropriate animals by oral gavage on Day 1, using a disposable catheter attached to a plastic syringe, as outlined in Table 9. The oral route of exposure was selected to provide readily available uric acid to the GI tract. Table 9.
  • Group 1 received 15N-UA dosing solution and 0.36 M sodium bicarbonate with SYN8669 (C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222); 3 copies each; cured of antibiotic resistance; ⁇ clb ⁇ dapA ⁇ ), and Group 2 received 15N-UA dosing solution and 0.36 M sodium bicarbonate with vehicle9.
  • SYN8669 C. californicus uricase (integrated) (SEQ ID NO: 219), E. tarda transporter (integrated) (SEQ ID NO: 222); 3 copies each; cured of antibiotic resistance; ⁇ clb ⁇ dapA ⁇ )
  • Group 2 received 15N-UA dosing solution and 0.36 M sodium bicarbonate with vehicle9.
  • Sample Collection X Sample to be collected. [0470] On Day 1, prior to dosing initiation, a clean collection pan was inserted to assist in urine and feces collection at room temperature. At conclusion of 6 hours post dose, the total amount of urine and feces was measured and recorded. [0471] For urine, an aliquot of 1 mL samples was collected in uniquely labeled clear polypropylene tube and immediately frozen on dry ice.
  • a second aliquot of approximately 100uL was collected in a 96-deep well plate (Corning Axygen® P-96-450R-C-S Storage 96-Well Assay Microplate with R- Bottom Wells, 96-Well x 500 ⁇ L, Clear PP, Sterile and CELLTREAT Foil Sealing Films) and immediately frozen on dry ice.
  • the total amount of feces produced by each animal was weighed. Depending on the amount produced, the entire feces sample or a single subsample (from cumulative and mixed fecal matter) of about 10 grams per animal (as available) was placed into a 50mL conical tube and immediately frozen on dry ice.
  • tarda transporter (integrated) (SEQ ID NO: 222); 3 copies each; cured of antibiotic resistance; ⁇ clb ⁇ dapA ⁇ ) was associated with a lowering (albeit not significant) of labeled uric acid at 1 h, and a corresponding increase in labeled allantoin at 1 and 2 h.
  • SYN8669 C. californicus uricase (integrated) (SEQ ID NO: 219)
  • tarda transporter (integrated) (SEQ ID NO: 222); 3 copies each; cured of antibiotic resistance; ⁇ clb ⁇ dapA ⁇ ) dosed at 1e11 was associated with a lowering of urinary labeled uric acid, and a concomitant increase in urinary output of labeled allantoin (FIGs.33D and 33E).
  • SYN8669 C. californicus uricase (integrated) (SEQ ID NO: 219), E.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Mycology (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des cellules hôtes qui comprennent une enzyme catabolique d'acide urique, par exemple, une enzyme dégradant l'acide urique, pour le traitement de maladies et de troubles associés à l'acide urique, y compris l'hyperuricémie et la goutte, chez un sujet. L'invention concerne en outre des compositions pharmaceutiques et des méthodes de traitement de troubles associés à l'acide urique, tels que l'hyperuricémie et la goutte.
PCT/US2022/075820 2021-09-01 2022-09-01 Cellules recombinantes pour le traitement de maladies associées à l'acide urique et leurs procédés d'utilisation WO2023034904A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2022340814A AU2022340814A1 (en) 2021-09-01 2022-09-01 Recombinant cells for treating diseases associated with uric acid and methods of use thereof

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US202163239517P 2021-09-01 2021-09-01
US63/239,517 2021-09-01
US202163251097P 2021-10-01 2021-10-01
US63/251,097 2021-10-01
US202263335411P 2022-04-27 2022-04-27
US63/335,411 2022-04-27

Publications (1)

Publication Number Publication Date
WO2023034904A1 true WO2023034904A1 (fr) 2023-03-09

Family

ID=85413113

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2022/075820 WO2023034904A1 (fr) 2021-09-01 2022-09-01 Cellules recombinantes pour le traitement de maladies associées à l'acide urique et leurs procédés d'utilisation

Country Status (2)

Country Link
AU (1) AU2022340814A1 (fr)
WO (1) WO2023034904A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7056713B1 (en) * 1998-08-06 2006-06-06 Duke University Urate oxidase
US20110171268A1 (en) * 2008-03-24 2011-07-14 Althea Technologies, Inc. Uricase compositions and methods of use
CN102220354A (zh) * 2011-05-01 2011-10-19 浙江大学 Microbacterium属细菌耐热尿酸氧化酶基因及其用途
CN110747157A (zh) * 2019-11-20 2020-02-04 深圳市诺维健生物技术有限责任公司 一种能在肠道中降解尿酸的工程益生菌及其制备方法与应用
WO2021173808A1 (fr) * 2020-02-25 2021-09-02 Synlogic Operating Company, Inc. Bactéries recombinées modifiées pour traiter des maladies associées à l'acide urique et leurs méthodes d'utilisation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7056713B1 (en) * 1998-08-06 2006-06-06 Duke University Urate oxidase
US20110171268A1 (en) * 2008-03-24 2011-07-14 Althea Technologies, Inc. Uricase compositions and methods of use
CN102220354A (zh) * 2011-05-01 2011-10-19 浙江大学 Microbacterium属细菌耐热尿酸氧化酶基因及其用途
CN110747157A (zh) * 2019-11-20 2020-02-04 深圳市诺维健生物技术有限责任公司 一种能在肠道中降解尿酸的工程益生菌及其制备方法与应用
WO2021173808A1 (fr) * 2020-02-25 2021-09-02 Synlogic Operating Company, Inc. Bactéries recombinées modifiées pour traiter des maladies associées à l'acide urique et leurs méthodes d'utilisation

Also Published As

Publication number Publication date
AU2022340814A1 (en) 2024-02-15

Similar Documents

Publication Publication Date Title
JP7245271B2 (ja) 高フェニルアラニン血症を低減させるように操作された細菌
EP3307870B1 (fr) Bactéries manipulées pour le traitement d'une maladie ou d'un trouble
AU2016356684C1 (en) Bacteria engineered to reduce hyperphenylalaninemia
US20230105474A1 (en) Recombinant bacteria engineered to treat diseases associated with uric acid and methods of use thereof
US20230090705A1 (en) Recombinant bacteria engineered to treat diseases associated with methionine metabolism and methods of use thereof
US20220362311A1 (en) Optimized bacteria engineered to treat disorders involving the catabolism of leucine, isoleucine, and/or valine
US20240102024A1 (en) Recombinant bacteria engineered to treat diseases associated with methionine metabolism and methods of use thereof
CA2996535A1 (fr) Bacteries modifiees pour traiter les troubles impliquant le catabolisme du propionate
WO2023034904A1 (fr) Cellules recombinantes pour le traitement de maladies associées à l'acide urique et leurs procédés d'utilisation
JP7494345B2 (ja) 高フェニルアラニン血症を低減させるように操作された細菌
US20230174926A1 (en) Bacteria engineered to treat disorders involving the catabolism of leucine

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22865793

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: AU2022340814

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2022340814

Country of ref document: AU

Date of ref document: 20220901

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE