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WO2025072960A2 - Procédés pour modulation de niveaux in vivo de sulfure d'hydrogène par des organismes modifiés - Google Patents

Procédés pour modulation de niveaux in vivo de sulfure d'hydrogène par des organismes modifiés Download PDF

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WO2025072960A2
WO2025072960A2 PCT/US2024/049315 US2024049315W WO2025072960A2 WO 2025072960 A2 WO2025072960 A2 WO 2025072960A2 US 2024049315 W US2024049315 W US 2024049315W WO 2025072960 A2 WO2025072960 A2 WO 2025072960A2
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engineered microorganism
seq
gene
promoter
set forth
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WO2025072960A3 (fr
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Ryan A. Koppes
Benjamin Woolston
Justin Hayes
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Northeastern University China
Northeastern University Boston
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Northeastern University China
Northeastern University Boston
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Definitions

  • H 2 S hydrogen sulfide
  • H 2 S s role as a therapeutic or toxic mediator in disease is contradictory, and appears to depend on concentration, disease target, and the route of administration.
  • H 2 S-based pharmaceutical drugs for gastrointestinal pain and inflammatory bowel disease (IBD) is being developed and engineered microbes as therapeutics for metabolic disorders such as phenylketonuria (PKU) are being developed.
  • IBD gastrointestinal pain and inflammatory bowel disease
  • PKU phenylketonuria
  • the present disclosure provides an engineered microorganism, comprising a first gene encoding a serine acetyltransferase; and a second gene encoding a cysteine synthase A.
  • the serine acetyltransferase has reduced L- cysteine feedback inhibition as compared to a wild-type serine acetyltransferase from the same bacterial subtype under the same conditions.
  • the serine acetyltransferase is mutated at positions 167 and/or 256.
  • the serine acetyltransferase comprises the amino acid sequence set forth in SEQ ID NO: 1 or 2.
  • the first gene is a CysE gene.
  • the CysE gene comprises the nucleic acid sequence set forth in SEQ ID NO: 2 or 4.
  • the cysteine synthase A comprises the amino acid sequence set forth in SEQ ID NO: 5.
  • the second gene is a CysK gene.
  • the CysK gene comprises the nucleic acid sequence set forth in SEQ ID NO: 6.
  • the engineered microorganism converts H 2 S to L-cysteine.
  • the engineered microorganism further comprises a third gene encoding a glutathione biosynthesis bifunctional protein or encoding a glutamate-cysteine ligase (ghsA) and a glutathione synthase (gshB).
  • the third gene encodes the glutathione biosynthesis bifunctional protein.
  • the glutathione biosynthesis bifunctional protein comprises the amino acid sequence set forth in SEQ ID NO: 7 or 9.
  • the third gene is a gshF (gshAB) gene.
  • the gshF gene comprises the nucleic acid sequence set forth in SEQ ID NO: 8 or 10.
  • the third gene encodes the glutamate-cysteine ligase and the glutathione synthase.
  • the glutamate-cysteine ligase comprises the amino acid sequence set forth in SEQ ID NO: 11.
  • the glutathione synthase comprises the amino acid sequence set forth in SEQ ID NO: 13.
  • the third gene comprises a gshA gene and a gshB gene.
  • 2 FH12512877.6 Attorney Docket No.: NEX-16225 the gshA gene comprises the nucleic acid sequence set forth in SEQ ID NO: 12.
  • the gshB gene comprises the nucleic acid sequence set forth in SEQ ID NO: 14.
  • the engineered microorganism converts H 2 S to L-cysteine, converts L-cysteine to gamma-L-glutamyl-L-cysteine, and converts gamma-L-glutamyl-L- cysteine to glutathione.
  • the engineered microorganism lacks native decR (DNA- binding transcriptional activator), iscS (cysteine desulfurase), yhaM (L-cysteine desulfidase) and/or yhaO (L-cysteine importer).
  • the decR comprises the amino acid sequence set forth in SEQ ID NO: 37 or encoded by the nucleic acid set forth in SEQ ID NO: 38.
  • the iscS comprises the amino acid sequence set forth in SEQ ID NO: 35 or encoded by the nucleic acid set forth in SEQ ID NO: 36.
  • the yhaM comprises the amino acid sequence set forth in SEQ ID NO: 25 or encoded by the nucleic acid set forth in SEQ ID NO: 26.
  • the yhaO comprises the amino acid sequence set forth in SEQ ID NO: 27 or encoded by the nucleic acid set forth in SEQ ID NO: 28.
  • the first gene, the second gene, the third gene, and/or the fourth gene is a heterologous or homologous gene.
  • the first gene, the second gene, and/or the third gene is operably linked to a promoter that is activated or repressed by an environmental cue.
  • the environmental cue is luminal H 2 S, reactive oxygen species (ROS), thiosulfate, a disease-relevant biomarker, or Interleukin (IL).
  • the promoter is a ROS-activated promoter.
  • the ROS-activated promoter is a oxyS promoter is an ROS-activated promoter.
  • the oxyS promoter is from E. coli.
  • the engineered microorganism expresses a transcription factor OyxR.
  • ROS activates the OxyR, which activates the native OxyS promoter from E. coli.
  • the OxyS comprises the nucleic acid sequence set forth in SEQ ID NO: 41.
  • the OxyR comprises the amino acid sequence set forth in SEQ ID NO: 39 or encoded by the nucleic acid set forth in SEQ ID NO: 40.
  • the promoter is a luminal H 2 S-activated promoter.
  • the luminal H 2 S-activated promoter is an IR9 promoter.
  • the bacterium is an auxotroph in a gene that is important for cellular growth.
  • the gene that is important for cellular growth is dapA, xylA, thyA, tyrA, glnA, cysE, metA, thrC, aspC, hisG, tyrB, proC, lysA, asnA, asnB, or any combination thereof.
  • the bacterium comprises a kill switch.
  • the present disclosure provides a pharmaceutically acceptable composition comprising the microorganism disclosed herein; and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable composition is formulated for oral or rectal administration.
  • the present disclosure provides a method of treating a disease associated with increased H 2 S levels, comprising administering to a subject in need thereof an effective amount of the engineered microorganism disclosed herein or the pharmaceutically acceptable composition disclosed herein.
  • the disease associated with increased H 2 S levels is a neurodegenerative disease, a myocardial injury, an ophthalmic disease, a gastrointestinal disorder, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), an ulcer, colorectal cancer, mental retardation, Down Syndrome, intestinal pain, H 2 S poisoning, an inflammatory disease, a cardiometabolic disease, Ethylmalonic encephalopathy, liver fibrosis, liver ischemia ⁇ reperfusion injury, hepatocellular carcinoma, hepatotoxicity, acute liver failure, osteoporosis, central nervous diseases like Alzheimer's 9 FH12512877.6 Attorney Docket No.: NEX-16225 disease, Parkinson's disease, Huntington's disease, and ischemic stroke, skin diseases
  • the present disclosure provides a method of treating a disease associated with decreased H 2 S levels, comprising administering to a subject in need thereof an effective amount of the engineered microorganism disclosed herein or the pharmaceutically acceptable composition disclosed herein.
  • the disease associated with decreased H 2 S levels neurodegenerative disease, a myocardial injury, an ophthalmic disease, a gastrointestinal disorder, an ulcer, colorectal cancer, intestinal pain, an inflammatory disease, a cardiometabolic disease, liver fibrosis, liver ischemia ⁇ reperfusion injury, hepatocellular carcinoma, hepatotoxicity, acute liver failure, osteoporosis, central nervous diseases like Alzheimer's disease, Parkinson’s disease, Huntington’s disease, and ischemic stroke, skin diseases such as burn, diabetic skin wound, psoriasis, systemic sclerosis, melanoma, pruritus.
  • Fig.5E shows schematic of GMPS co-culture with engineered microbes and end-point analyses.
  • Figs.6A-6C show H 2 S production across the gut physiological range with engineered strains in Hungate tubes.
  • Figs.6A-6C shows H 2 S production quantified in Hungate tubes over four hours, starting OD6000.4. Samples were taken while maintaining a closed system.
  • Fig.6A shows that the three knockout strains were compared to wild type MG1655 in PBS++ (non-193 growth media) or Fig.6B shows in M9 minimal media (growth media).
  • Fig.6A shows that the three knockout strains were compared to wild type MG1655 in PBS++ (non-193 growth media) or Fig.6B shows in M9 minimal media (growth media).
  • Fig.6A shows that the three knockout strains were compared to wild type MG1655 in PBS++ (non-19
  • FIG. 6C shows engineered strains tested in PBS++ (non-growth media).
  • the blue-shaded region represents the estimated gut physiological range (300-3,400 ⁇ M H 2 S).
  • Error bars represent SD, and bars represent the mean value.
  • Figs.7A-7C show titratable microbial H 2 S production in the GMPS.
  • Fig.7A shows schematic of the experimental set-up.
  • Figs.8A-8E show characterization of microbial colonization and metabolism in GMPS.
  • Fig.8A shows schematic of the experimental set-up. Glucose diffuses into the apical channel and is metabolized into acetate by E. coli. Cysteine was perfused through the chip, and the high-sulfide strain produced H 2 S.
  • Figs.8C-8D show representative images of the co-culture.400X live confocal images, post-processed with Gaussian smoothing.
  • Fig.8C shows high-sulfide strain expressing RFP in GMPS after two days of co- culture.
  • Fig.8D shows Z-stack cross-sectional image of the high-sulfide strain expressing RFP and Caco-2 cell stained nuclei in the GMPS after one day of co-culture.
  • Fig.8E shows representative image of the co-culture.100X live image of RFP expressing E. coli MG1655 with RFP and DIC filters captured with an inverted microscope. Error bars represent SD, and bars represent the mean value. Figure created with the World Wide Web at Biorender.com.
  • Figs.9A-9D show microbially derived H 2 S affects Caco-2 cells in a concentration- dependent manner.
  • Fig.9A shows schematic of the experimental set-up. Cysteine was perfused through the chip, and the engineered strains produced H 2 S. Paracellular diffusion into the basal channel was quantified using Lucifer Yellow, and Papp was determined. Microbially derived or exogenous H 2 S was metabolized into thiosulfate by Caco-2 cells and was detected with HPLC.
  • Fig.9B shows Papp (cm/s) was determined by collecting basal effluent before (hour -4) and during treatment conditions (hours 18 and 42). Dots indicate samples taken from the same biological replicate for identifying trends in permeability before and after treatment.
  • Fig.9C shows apical effluent was collected and analyzed for thiosulfate via HPLC, data shown from hour 42.
  • Fig.9C shows corresponding apical H 2 S production levels at hour 44.
  • Fig.9D shows Caco-2 RNA was extracted after two days of treatment. (left) Fold change expression of GADD45a mRNA levels (reference gene GAPDH). Fold change expression was normalized to the Control.
  • Fig.10A shows OD600 measurements of strains in M9 minimal media and Fig.10B shows in M9 minimal media with 5 mM cysteine.
  • Fig.14 shows example of H 2 S standard curves in PBS++ and DMEM. Standard curves were made according to the methylene blue assay.
  • Fig.16 shows schematic of CTX103 in vivo (left) and the engineered metabolic pathway (right). Briefly, GSH is transported into the cell and degraded through two reactions to Cys. Cys is rapidly converted to sulfide which diffuses out of the cell. The cysteine transporter, yhaO, is also expressed to scavenge any Cys that may be exported from the cell. Figure created with the World Wide Web at Biorender.com. Fig.17 shows cells were induced overnight, pelleted, and resuspended in minimal glucose media (M9) under anaerobic conditions.1 mM GSH was added to the culture.
  • M9 minimal glucose media
  • Fig.19 shows schematic of CTX102 in vivo (left) and the engineered metabolic pathway (right). Briefly, l-serine and acetyl-CoA are generated from the TCA cycle.
  • cysE** converts these into o-acetyl-l-serine, which reacts with H 2 S, catalyzed by cysK, to synthesize cysteine (cys).
  • Cys is exported from the cell through passive and active transporters or converted to GSH by ligating Cys, glycine (Gly), and glutamate (Glut) catalyzed by gshF which requires 2 ATP.
  • Fig.23 shows schematic of CTX111/CTX112 in vivo (left) and the engineered metabolic pathway (right). Briefly, H 2 S oxidized by the SQR enzyme, and these electrons are integrated into the electron transport chain via the quinone pool (CoQ). The sulfur atom is assimilated into glutathione persulfide and other polysulfides. Figure created with the World Wide Web at Biorender.com.
  • Fig.28 shows that cells were induced to express the thiosulfate sensing system with increasing amounts of thiosulfate, pelleted, and resuspended in LB media under aerobic conditions.
  • DETAILED DESCRIPTION Strains were developed to modulate intestinal H 2 S levels. Modulation of H 2 S levels is split into two modules: consumption and production. In the consumption module, we engineered two synthetic pathways for sulfide sequestration. The first pathway sequesters H 2 S into glutathione.
  • cysE and cysK from Escherichia coli (E. coli) were cloned and expressed in a plasmid.
  • cysE was mutated in two places (AA167 and AA256) to prevent feedback inhibition by L-cysteine (the product).
  • cysE transforms serine and acetyl-CoA into o-acetyl-serine (OAS), and cysK transforms H 2 S and OAS into L-cysteine.
  • OFS o-acetyl-serine
  • cysK transforms H 2 S and OAS into L-cysteine.
  • NEX-16225 the two different pathways to synthesize glutathione from cysteine.
  • gshA which ligates cysteine and glutamate into gamma-L-glutamyl-L- cysteine
  • gshB which ligates L-glutamate and into gamma-L-glutamyl-L-cysteine to form glutathione.
  • the bifunctional gshF gene from Streptococcus thermophilus was cloned which converts L-cysteine, L-glutamate, and glycine into glutathione.
  • luminal H 2 S is consumed by the microbe and transformed into the intermediate L-cysteine, which is then converted to glutathione as a stable sulfur sink.
  • Storing sulfur in the form of glutathione has two advantages: i) It is a known anti-oxidant and may benefit disease; and ii) Glutathione is stable in the human gut microbiome.
  • the second pathway sequesters H 2 S by oxidation by a sulfide:quinone reductase (SQR) from Wolinella succinogenes or Rhodabacter capsulatus.
  • SQR quinone reductase
  • H 2 S is oxidized and the electrons are transferred to the quinone pool and electron transport chain, and the sulfur atom is assimilated into glutathione persulfide, polysulfide compounds, or elemental sulfur.
  • SQR quinone reductase
  • the first pathway glutathione is transported into the cell and broken down by pepT and ggt (E. coli homologous enzymes) into L-cysteinylglycine, and further broken into L-cysteine by pepB (E. coli homologous enzyme).
  • L-cysteine is converted to sulfide by the native desulfidase, yhaM, and is passively exported to the intestinal lumen.
  • the native L- cysteine transporter, yhaO is expressed to sequester environmental L-cysteine for conversion to sulfide. All genes are native to E. coli.
  • the second pathway uses a sulfite reductase to convert sulfite to sulfide. The E.
  • coli native cysI and cysJ subunits form the CysIJ sulfite reductase and convert sulfite to sulfide.
  • the cysG gene is also overexpressed to synthesize siroheme required for cysIJ.
  • Genes from both the production and consumption modules can be activated or repressed by environmental cues, such as luminal H 2 S, reactive oxygen species (ROS), thiosulfate, or disease biomarkers such as the interleukins (IL) and pro-inflammatory compounds.
  • ROS reactive oxygen species
  • thiosulfate or disease biomarkers
  • IL interleukins
  • pro-inflammatory cascades in inflammatory bowel disease (IBD) result in high levels of ROS, which can trigger ROS-activated promoters driving the expression of downstream genes.
  • a thiosulfate-responsive system was assembled to sense environmental thiosulfate and respond by degrading H 2 S.
  • the thsS a membrane-bound thiosulfate sensor which phosphorylates the transcription factor, thsR, which binds to 16 FH12512877.6
  • NEX-16225 the phsA342 promoter to drive gene expression from Shewanella halifaxensis. This system was adapted into E.
  • the environmental cue can be ROS, using a ROS-activated promoter such as the oxyR and oxyS gene-promoter from E. coli. High ROS levels are associated with a pro- inflammatory environment and can be used as a signal to drive gene expression for H 2 S sequestration pathways.
  • the environmental cue can be H 2 S, using a luminal H 2 S-activated promoter, such as native promoter, IR9, from B. licheniformis.
  • a luminal H 2 S-activated promoter such as native promoter, IR9, from B. licheniformis.
  • NreB senses H 2 S and phosphorylates the transcription factor, NreC, which binds to a native promoter from B. licheniformis.
  • the environmental cue can be pro-inflammatory biomarkers such as interleukins (IL) or other chemokines which drive a signaling cascade and drive gene expression of H 2 S production or consumption pathways.
  • IL interleukins
  • NF- ⁇ B transcription factors p65 (RelA), RelB, c-Rel, p105p50 (NF- ⁇ B1), and p100/52 (NF- ⁇ B2)
  • TNF tumor necrosis factor
  • R A or G
  • N A, C, G, or T
  • W A or T
  • Y C or T.
  • the NF- ⁇ B transcription factors regulate hundreds of genes.
  • the consensus binding sequence can be used to bind activated NF- ⁇ B transcription factors to drive expression of H 2 S consumption or production pathways in microbes.
  • the disclosed microbes provide sense-and-respond therapeutic platform. Most therapeutics are 'static', meaning they cannot respond to the surrounding environment. Our microbes can sense the intestinal environment and toggle between consumption and production of sulfur species. The gut is a heterogeneous environment (large patient-to-patient variation) which gives our microbial platform an advantage over traditional one-dimensional therapies, such as chemical sulfide donors.
  • the disclosed microbes provide recycling of a toxic metabolite (H 2 S) to a therapeutic metabolite (glutathione). 17 FH12512877.6 Attorney Docket No.: NEX-16225
  • the disclosed microbes provide a microbial platform that can be genetically modified for different applications and disease targets.
  • Microbial-based consumption/delivery of sulfur compounds is advantageous over chemical donors because of dynamic control enabled by gene expression in the microbes. This can be in response to environmental factors (ROS, thiosulfate, pro-inflammatory levels, or H 2 S levels).
  • ROS environmental factors
  • thiosulfate thiosulfate
  • pro-inflammatory levels or H 2 S levels
  • chemical sulfide chelators or sulfide-releasing compounds are not dynamic nor targeted to specific regions in the gut. This may be advantageous because there is large patient-to-patient variation in many diseases.
  • the disclosed engineered bacteria sequester H 2 S into intracellular and extracellular sulfur compounds that are inaccessible to the native human gut microbiome for H 2 S production. This will lead to a stable and reliable reduction in intestinal sulfide for therapeutic applications.
  • Potential uses include: Hydrogen sulfide exerts its effects in a concentration-dependent manner, and therefore can exacerbate diseases when too much or too little of the compound is present in a biological setting.
  • the disclosed microbes provide consumption of H 2 S in neurodegenerative disease, a myocardial injury, an ophthalmic disease, a gastrointestinal disorder, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), an ulcer, colorectal cancer, mental retardation, Down Syndrome, intestinal pain, H 2 S poisoning, an inflammatory disease, a cardiometabolic 18 FH12512877.6
  • High H 2 S levels are associated with exaggerated disease.
  • Mental retardation in Down Syndrome may be caused by high H 2 S levels in the blood and brain.
  • H 2 S-producing enzymes in people with DS which may be considered a metabolic disorder.
  • Our engineered microbes sequester excess H 2 S via the intestine. H 2 S passively diffuses through tissue, and systemic H 2 S levels could be reduced via this mechanism.
  • Synlogic’s SYNB1934 is in Phase III trials and sequesters systemic phenylalanine via the gut, effectively reducing serum phenylalanine levels for PKU disease treatment. Similar to the connection between DS and H 2 S, excess systemic phenylalanine induces brain damage in patients with PKU.
  • the disclosed microbes provide production of H 2 S in neurodegenerative disease, a myocardial injury, an ophthalmic disease, a gastrointestinal disorder, an ulcer, colorectal cancer, intestinal pain, an inflammatory disease, a cardiometabolic disease, liver fibrosis, liver ischemia ⁇ reperfusion injury, hepatocellular carcinoma, hepatotoxicity, acute liver failure, osteoporosis, central nervous diseases like Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and ischemic stroke, skin diseases such as burn, diabetic skin wound, psoriasis, systemic sclerosis, melanoma, pruritus, or diabetes mellitus. (Low H 2 S levels are associated with exaggerated disease).
  • the disclosed microbes provide delivery of antioxidants (H 2 S, L-cysteine, and glutathione) to sources of intestinal inflammation/damage (IBD, ulcers, colorectal cancer).
  • the disclosed microbes provide treatments for inflammatory bowel disease (IBD), H 2 S- mediated mental retardation, and/or intestinal pain relief.
  • IBD inflammatory bowel disease
  • H 2 S- mediated mental retardation and/or intestinal pain relief.
  • the disclosed microbes provide pain relief in the gut.
  • the disclosed microbes reduce bowel movements in the gut for treating diarrhea and symptomology of various gut diseases.
  • the disclosed microbes provide treatment for H 2 S poisoning (usually caused by workplace exposure).
  • Performance advantages include: The dynamic control aspect of the microbes enables the technology to toggle between production and consumption, which may enhance its efficacy and the number of patients it is helpful for.
  • the engineered microbes are generally regarded as safe and provide unprecedented control over intestinal metabolites.
  • Provided herein are microorganisms, systems and methods for modulating H 2 S.
  • the present disclosure provides genetically engineered microorganisms capable of consuming H 2 S, converting H 2 S to L-cysteine and optionally converting L- cysteine to glutathione, or converting H 2 S to persulfide and polysulfide compounds.
  • the present disclosure provides genetically engineered microorganisms capable of producing H 2 S, converting glutathione to L-cysteine and H 2 S, and converting sulfite into sulfide.
  • Ranges are stated in shorthand to avoid having to set out at length and describe each and every value within the range. Therefore, when ranges are stated for a value, any 20 FH12512877.6 Attorney Docket No.: NEX-16225 appropriate value within the range can be selected, and these values include the upper value and the lower value of the range. For example, a range of two to thirty represents the terminal values of two and thirty, as well as the intermediate values between two to thirty, and all intermediate ranges encompassed within two to thirty, such as two to five, two to eight, two to ten, etc.
  • the term “genetic modification” as used herein refers to altering the genomic DNA in a microorganism.
  • a genetic modification alters the expression and/or activity of a protein encoded by the altered gene.
  • a genetic modification encompasses a “variant”, which is a gene or protein sequence that deviates from a reference gene or protein, as further detailed below.
  • microorganism refers to prokaryote or eukaryote microorganisms capable of oligosaccharides production or utilization with or without modifications.
  • parental microorganism refers to a microorganism that is manipulated to produce a genetically modified microorganism.
  • a gene is mutated in a microorganism by one or more genetic modifications
  • the microorganism being modified is a parental microorganism of the microorganism carrying the one or more genetic modifications.
  • the term “gene” includes the coding region of the gene as well as the upstream and downstream regulatory regions.
  • the upstream regulatory region includes sequences comprising the promoter region of the gene.
  • the downstream regulatory region includes sequences comprising the terminator region. Other sequences may be present in the upstream and downstream regulatory regions.
  • a gene is represented herein in small caps and italicized format of the name of the gene, whereas a protein is represented in all caps and non-italicized format of the name of the protein.
  • cdt-1 represents a gene encoding the CDT-1 protein
  • CDT-1 non-italicized and all caps
  • sequence identity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% to a reference sequence refers to a comparison made between two sequences, preferably using the BLAST algorithm. Algorithms for comparisons between two protein sequences that use protein structural information, such as sequence threading or 3D- 1D profiles, are also known in the field.
  • a “variant” is a gene or protein sequence that deviates from a reference gene or protein.
  • isoform also refer to “variant” forms of a gene or a protein.
  • the variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine.
  • a 21 FH12512877.6 Attorney Docket No.: NEX-16225 variant may have “nonconservative” changes, e.g., replacement of a glycine with a tryptophan.
  • Analogous minor variations may also include amino acid deletions or insertions, or both.
  • An endogenous sequence is “native” to, i.e., indigenous to, the microorganism.
  • the term “mutation” refers to genetic modification to a gene including modifications to the open reading frame, upstream regulatory region, and/or downstream regulatory region.
  • a heterologous host cell for a nucleic acid sequence refers to a cell that does not naturally contain the nucleic acid sequence.
  • a “chimeric nucleic acid” comprises a first nucleotide sequence linked to a second nucleotide sequence, wherein the second nucleotide sequence is different from the sequence which is associated with the first nucleotide sequence in cells in which the first nucleotide sequence occurs naturally.
  • a constitutive promoter expresses an operably linked gene when RNA polymerase holoenzyme is available. Expression of a gene under the control of a constitutive promoter does not depend on the presence of an inducer.
  • An inducible promoter expresses an operably linked gene only in the presence of an inducer.
  • An inducer activates the transcription machinery that induces the expression of a gene operably linked to an inducible promoter.
  • the term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition.
  • prevention of seizures includes, for example, reducing the number of seizures in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable lesions in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.
  • 22 FH12512877.6 Attorney Docket No.: NEX-16225
  • subject refers to a mammal, including, but not limited to, a human or non- human mammal, such as a bovine, equine, canine, ovine, or feline.
  • administering means providing a therapeutic agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering.
  • the means of providing a therapeutic agent are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, intraaural administration, intracerebral administration, rectal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent.
  • a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition.
  • the chip was engineered to maintain H 2 S gas tension and enabled visualization of co-culture in real-time with confocal microscopy.
  • Engineered strains colonized the chip and were metabolically active for two days, during which they produced H 2 S across a sixteen-fold range and induced changes in host gene expression and metabolism in an H 2 S concentration-dependent manner.
  • Colonic H 2 S is produced by sulfate-reducing bacteria (SRB) and cysteine-degrading bacteria, with the latter being more abundant in the microbiome.
  • SRB sulfate-reducing bacteria
  • cysteine-degrading bacteria Several lines of evidence support a role for H 2 S as a toxic mediator of inflammation.
  • IBD inflammatory bowel disease
  • UC ulcerative colitis
  • H 2 S donors induce cell cycle arrest in immortalized intestinal epithelial Caco-2 cells and affect proliferation of the immortalized intestinal epithelial cell line, HCT116, in a concentration-dependent manner.
  • mice colonized with a human microbiota are an advancement, but may not capture specific interactions in the human gut that have arisen from the co-evolution of the 26 FH12512877.6 Attorney Docket No.: NEX-16225 host and microbiota.
  • NEX-16225 host and microbiota For static microbial/human cell co-cultures, the lack of fluid flow leads to microbial overgrowth in co-cultures, so only short time scales can be explored.
  • in vitro flow systems such as the gut-on-a-chip (GoC) are emerging as excellent models for studying host-microbe interactions. They are easier to probe than animal models, and recreate certain organ level functions like transport, digestion, and metabolism better than static Transwell and intestinal organoid cultures.
  • RT-qPCR analysis of epithelial cells showed an H 2 S concentration-dependent effect on host expression of GADD45a, a gene involved in DNA damage and cell cycle arrest known to be upregulated in the presence of H 2 S. Additionally, metabolic analysis of co-culture effluent from the lumen channel showed host oxidation of microbial H 2 S into thiosulfate. Finally, our platform allows for live visualization of colonization with confocal fluorescence microscopy. Overall, we have validated a powerful platform combining synthetic biology and GMPSs for investigating the role of H 2 S in the gut, which supports fundamental discoveries in human health. Discussion Investigating the mechanistic role of microbially derived metabolites on host biology remains experimentally difficult.
  • Na 2 S is not a natural sulfide source in the human gut, and HPLC analysis showed different thiosulfate profiles when compared to cysteine-derived sulfide, which is a major production pathway in the human gut. Additionally, diffusion of H 2 S to the epithelial cell surface may depend on gut biogeography.
  • the constant fluid flow in the system promotes the stable co- culture of microbes and host epithelial cells, enabling longer duration experiments without the problems of microbial overgrowth.
  • This platform is not limited to gaseous metabolites. Synthetic biology allows for plug-and-play of different genetic elements to study other metabolites, such as neurotransmitters. Gut microbes affect the brain’s neurotransmitter pool by modulating precursors in the gut that communicate via the gut-brain-axis . More broadly, translating synthetic probiotics from test tubes to the clinic will require extensive validation and additional engineering to ensure robust performance in the complex 30 FH12512877.6 Attorney Docket No.: NEX-16225 gut environment. Incorporating the GMPS into the design-build-test-learn cycle for smart probiotics could help identify factors underlying strain stability, performance, and robustness at lower cost and with more fidelity than mouse studies.
  • GoC systems have accurately predicted the in vivo strain activity of a clinical biotherapeutic, supporting their role as an orthogonal pre-clinical model.
  • a powerful platform combining synthetic biology and gas tension-retaining GMPSs for investigating the role of gaseous mediators in the human intestine.
  • This platform enables precise manipulation of the gut metabolome, which we leveraged to understand the concentration-dependent effects of H 2 S on human gut epithelial cells.
  • This platform will allow researchers to experimentally address host-microbe hypotheses that are infeasible with current animal and in vitro models.
  • Heterologous expression means that a protein is experimentally put into a cell that does not normally make (i.e., express) that protein.
  • Heterologous meaning ‘derived from a different organism’ refers to the fact that often the transferred protein was initially cloned from or derived from a different cell type or a different species from the recipient.
  • the protein itself is not transferred, but instead the genetic material coding for the protein (the complementary DNA or cDNA) is added to the recipient cell.
  • Methods for transferring foreign genetic material into a recipient cell include transfection and transduction.
  • H 2 S Consumption Provided herein are microorganisms, systems and methods for modulating H 2 S.
  • the present disclosure provides genetically engineered microorganisms capable of consuming H 2 S.
  • the microorganism described herein can converts H 2 S to L-cysteine and optionally converts L-cysteine to glutathione.
  • the microorganism is genetically engineered to express a serine acetyltransferase, or homologs and variants thereof.
  • CysE Serine acetyltransferase carries out the first step in the pathway of cysteine biosynthesis, converting L-serine into O-acetyl-L-serine.
  • serine acetyltransferase is provided by the sequence of SEQ ID NO: 1, which is serine acetyltransferase from Escherichia coli. Homologues of serine acetyltransferase from microorganisms other than Escherichia coli can be used in the microorganisms and methods described herein.
  • Non-limiting examples of the homologs of 31 FH12512877.6 Attorney Docket No.: NEX-16225 serine acetyltransferase in the instant invention are represented by UniProt entries: P0A9D4, Q06750, P95231, A0A120HUS7, P29847, Q8CTU2, P71405, P77985, P67765, Q6GBV9, P57162, P67764, P0A9D7, Q89B11, Q5HIE6, Q6GJE0, P67766, Q5HRM4, P32003, P43886, Q65PC9, Q9ZK14, Q56002, P74089, A8F961, P0A9D5, and P0A9D6.
  • serine acetyltransferase is provided by the amino acid sequence of SEQ ID NO: 1 or 3, which is serine acetyltransferase from Escherichia coli and encoded by the nucleic acid sequence of SEQ ID NO: 2 or 4.
  • SEQ ID NO: 1 or 3 is serine acetyltransferase from Escherichia coli and encoded by the nucleic acid sequence of SEQ ID NO: 2 or 4.
  • the UniProt entries listed herein are incorporated by reference in their entireties.
  • CysK Cysteine synthase A carries out the second step in the pathway of cysteine biosynthesis, converting O-acetyl-serine into L-cysteine.
  • An example of cysteine synthase A is provided by the sequence of SEQ ID NO: 5, which is cysteine synthase A from Escherichia coli. Homologues of serine acetyltransferase from microorganisms other than Escherichia coli can be used in the microorganisms and 33 FH12512877.6 Attorney Docket No.: NEX-16225 methods described herein.
  • Non-limiting examples of the homologs of serine acetyltransferase in the instant invention are represented by UniProt entries: P0ABK5, P37887, P0A1E3, and P9WP55.
  • An example of cysteine synthase A is provided by the amino acid sequence of SEQ ID NO: 5, which is cysteine synthase A from Escherichia coli and encoded by the nucleic acid sequence of SEQ ID NO: 6.
  • the UniProt entries listed herein are incorporated by reference in their entireties.
  • GshF is a bifunctional enzyme that acts as both a glutamate-cysteine ligase and a glutathione synthase.
  • An example of a glutathione biosynthesis bifunctional protein (glutamate-cysteine ligase and a glutathione synthase) is provided by the sequence of SEQ ID NO: 7 or 9, which is a glutathione biosynthesis bifunctional protein from Streptococcus thermophilus. Homologues of glutathione biosynthesis bifunctional protein from microorganisms other than Streptococcus thermophilus can be used in the microorganisms and methods described herein.
  • Non-limiting examples of the homologs of serine glutathione biosynthesis bifunctional protein in the instant invention are represented by UniProt entries: Q5M3J8, Q8DXM9, Q6ANW2, Q9CM00, Q8E399, Q5M3J8, Q65RX0, A0AMA3, Q5LYY5, Q8XK30, Q8Y3R3, Q926X7, Q71VZ1, Q82ZG8, Q8DW15, Q88UW5, A0A0A7RQH3, R9SL56, A0A0D0Y7W0, A0A0U3CHT1, D3E133, A0A140D6V9, A0A7X2T160, A0A427RZV8, A0A8B3TED9, A0A3S4XIR0, A0A1V4B0F6, A0A377IBZ0, A0A377JF93, A0A0F5RHX7, A0A378MUJ
  • glutathione biosynthesis bifunctional protein is provided by the amino acid sequence of SEQ ID NO: 7 or 9, which is a glutathione biosynthesis bifunctional protein from Streptococcus thermophilus and encoded by the nucleic acid sequence of SEQ ID NO: 8 or 10.
  • the UniProt entries listed herein are incorporated by reference in their entireties. 35 FH12512877.6 Attorney Docket No.: NEX-16225 gshF (S. thermophilus, codon harmonized for E.
  • thermophilus codon harmonized for E. coli
  • 1 atgaacctgc ggcatattat taaacagaac catttagaac tgctgttcca 51 gcaaggatcg ttcggattag aaaagagag ccaacgtgta cgccacgacg 101 gctcggtggt gactagcgcc cacccgaagg cgttcggcaa tcgttcgttt 151 catccgtata tacagacgga tttcgccgag agccaattag aactgattac 201 tccgcgaac aagaaattag aagatacgtt tcgctggtta cagactatcc 251 atgaagtggt tggcggacc ctgcggaag acgaattcatcatacc c
  • thermophilus 1 MTLNQLLQKL EATSPILQAN FGIERESLRV DRQGQLVHTP HPSCLGARSF 37 FH12512877.6
  • GQTDMIAFKN ALYLKLAQNY LRYRWVITYL FGASPIAEQG 201
  • FFDQEVPEPV RSFRNSDHGY VNKEEIQVSF VSLEDYVSAI ETYIEQGDLN 251
  • AEKEFYSAVR FRGQKVNRSF LDKGITYLEF RNFDLNPFER IGISQTTMDT 301 VHLLILAFLW LDSPENVDQA LAQGHALNEK
  • thermophilus 1 ATGACATTAA ACCAACTTCT TCAAAAACTG GAAGCTACCA GCCCTATTCT 51 CCAAGCTAAT TTTGGAATCG AGCGCGAGAG TCTACGTGTC GATAGGCAAG 101 GACAACTGGT GCATACACCT CACCCATCCT GTCTAGGAGC TCGTAGTTTC 151 CACCCCTATA TTCAGACTGA TTTTTGCGAG TTTCAGATGG AACTCATCAC 201 ACCAGTTGCC AAATCTACTA CTGAGGCTCG CCGATTTCTG GGAGCTATTA 251 CTGATGTAGC TGGCCGCTCTCT ATTGCTACAG ACGAGGTTCT CTGGCCTTTA 301 TCCATGCCAC CTCGTCTAAA GGCAGAGGAG ATTCAAGTTG CTCAACTGGA 351 AAATGACTTC GAACGCCATT ATCGTAACTA TTTGGCTGAA AAATACGGAA 401 CTAAACTACA AGCTATCTCA GGTATCCACT ATAATAATATGGA
  • GshA and GshB is a glutamate-cysteine ligase and a glutathione synthetase, respectively. 39 FH12512877.6 Attorney Docket No.: NEX-16225
  • An example of a glutamate-cysteine ligase or a glutathione synthase is provided by the sequence of SEQ ID NO: 11 or 13, respectively, which is a glutamate-cysteine ligase or a glutathione synthase from E. Coli. Homologues of glutamate-cysteine ligase or a glutathione synthase from microorganisms other than E.
  • Coli can be used in the microorganisms and methods described herein.
  • Non-limiting examples of the homologs of a glutamate-cysteine ligase in the instant invention are represented by UniProt entries: P9WPK7, Q54PC2, P9WPK6, P0A6W9, A0R5N1, Q8D2V5, A0A240ERG2, A0A480APB4, A0A379VLT6, A0A0F7KSP3, A0A1E3H7E4, A0A4P1KG84, A0A369QAZ4, Q926D5, A0A368A3B0, F7X981, P61379, Q5V3Q5, B5RDE8, Q7N7A4, Q7W3F2, A9HYE0, O68838, Q2NVK9, Q3IEB7, Q66E64, Q87LS2, P0A6X0, and B4EUV9.
  • Non-limiting examples of the homologs of a glutathione synthase in the instant invention are represented by UniProt entries: P04425, Q8D335, Q8DKF1, Q54E83, Q7U3W8, Q9ABS9, Q89WL0, Q7NF44, Q87LK1, P59495, Q9KUP7, Q83Q91, Q9PC29, P57612, P58580, Q7MHK1, Q7TUG9, Q82V16, Q8PHZ5, Q9I697, Q9JYJ3, Q7W910, Q8FXW6, Q8P6P1, Q9JTJ6, P58579, Q87D42, Q8DCA9, Q7NQ65, Q7TVB0, and Q87VA4.
  • glutamate-cysteine ligase is provided by the amino acid sequence of SEQ ID NO: 11, which is a glutamate-cysteine ligase from E. coli and encoded by the nucleic acid sequence of SEQ ID NO: 12.
  • An example of a glutathione synthase is provided by the amino acid sequence of SEQ ID NO: 13, which is a glutathione synthase from E. coli and encoded by the nucleic acid sequence of SEQ ID NO: 14.
  • the UniProt entries listed herein are incorporated by reference in their entireties.
  • the engineered microorganism expresses SQR to sequester and store the 42 FH12512877.6 Attorney Docket No.: NEX-16225 H 2 S by-product (e.g., persulfides, polysulfides, elemental sulfur, or other sulfide by-products) intracellularly for the purpose of preventing the release of the sulfur/recycling of the sulfur back to H2S by the native microbiota and or host cells.
  • SQOR The protein encoded by this gene may function in mitochondria to catalyze the conversion of sulfide to persulfides, thereby decreasing toxic concentrations of sulfide.
  • SQOR sulfide quinone reductase
  • SEQ ID NO: 15 or 17 is sulfide quinone reductase (SQOR) enzyme from Rhodobacter capsulatus or from Wolinella succinogenes, respectively.
  • SQOR sulfide quinone reductase
  • Homologues of sulfide quinone reductase (SQOR) enzyme from microorganisms other than Rhodobacter capsulatus or Wolinella succinogenes can be used in the microorganisms and methods described herein.
  • Non-limiting examples of the homologs of sulfide quinone reductase (SQOR) enzyme in the instant invention are represented by UniProt entries: Q52722, Q9Y6N5, Q9R112, Q54DK1, Q8NFU3, Q12305, A0A812UN59, A0A813AHA6, B0BMT9, A0A670YA51, A0A6J0TFA8, A0A6P9D5C0, A0A8C7E7R8, A0A8D0ED51, A0A9F2QUP7, A0A670JN21, A0A6J1UR84, A0A8C5RDU1, A0AA97KN11, A0A8D0L849, A0A8I3Q5D3, F1QYT2, A0A1L8H0P4, A0A7K4Q091, 0A7K4RTG5, A0A7K4ZCV4, A0A7K5GD
  • SQOR sulfide quinone reductase
  • the present disclosure provides genetically engineered microorganisms capable of producing H 2 S.
  • the microorganism described herein can converts L- cysteine to H 2 S and optionally converts glutathione to L-cysteine.
  • the microorganism is genetically engineered to express a peptidase T, or homologs and variants thereof.
  • PepT is an enzyme that removes the N-terminal amino acid from tripeptides.
  • An example of peptidase T is provided by the sequence of SEQ ID NO: 19, which is peptidase T from Escherichia coli.
  • Homologues of peptidase T from microorganisms other than Escherichia coli can be used in the microorganisms and methods described herein.
  • Non- limiting examples of the homologs of peptidase T in the instant invention are represented by UniProt entries: P29745, P0C2T7, Q76HM5, P26311, Q9L4G1, Q84BV2, Q76HM7, P81207, Q21219, Q81WU4, Q835J5, Q6D4E3, P55179, Q38X92, A9VR36, Q5WK52, A7ZAB2, B5QXA9, P65805, Q4L4G8, Q6GIP8, Q8Y6B1, Q9CP05, A2RF89, B8ZPG1, P58794, Q64WS4, Q6GB87, Q8XPD8, Q92AM8, B7VSC8, B5BAE8, P65806, Q5HHS7, Q5LFT7, Q5PMK2, Q
  • peptidase T is provided by the amino acid sequence of SEQ ID NO: 19, which is peptidase T from Escherichia coli and encoded by the nucleic acid sequence of SEQ ID NO: 20. 46 FH12512877.6 Attorney Docket No.: NEX-16225 The UniProt entries listed herein are incorporated by reference in their entireties. pepT (E.
  • PepB is a metallopeptidase belonging to the M17 family and is classified as a leucine aminopeptidase (LAP) and has cysteinylglycine dipeptidase activity based on sequence and activity.
  • LAP leucine aminopeptidase
  • An example of peptidase B is provided by the sequence of SEQ ID NO: 21, which is peptidase B from Escherichia coli. Homologues of peptidase B from microorganisms other than Escherichia coli can be used in the microorganisms and methods described herein.
  • Non- limiting examples of the homologs of peptidase B in the instant invention are represented by UniProt entries: P37095, Q9RF52, Q53778, Q6D266, Q8E0C9, A7MU37, B7VJT3, Q667Y8, Q7MNF5, Q9CM16, A8A329, B1LNH8, Q1C5H7, Q8DEZ4, Q8Z4N3, A4TMU7, B5FR78, B6I595, Q31XW7, Q32D41, Q57LH5, Q9KTX5, B5R595, Q7N231, Q87S21, A9N1Y3, A9R811, B4T0R5, C0PYL4, A9MHK1, B1IWD8, B1JRZ6, B2K9R0, C6DBI4, Q83QK5, B1XAZ8, B7N6B0, C4ZX98, Q1R8K9, Q3YZ29, Q1CKA2, P
  • peptidase B is provided by the amino acid sequence of SEQ ID NO: 21, which is peptidase B from Escherichia coli and encoded by the nucleic acid sequence of SEQ ID NO: 22.
  • the UniProt entries listed herein are incorporated by reference in their entireties. pepB (E.
  • NEX-16225 ggt is an enzyme that hydrolyzes the gamma-glutamyl bond of extracellular reduced and oxidized glutathione, initiating their cleavage into glutamate, cysteine (cystine) and glycine.
  • glutathione hydrolase is provided by the sequence of SEQ ID NO: 23, which is glutathione hydrolase from Escherichia coli. Homologues of glutathione hydrolase from microorganisms other than Escherichia coli can be used in the microorganisms and methods described herein.
  • Non-limiting examples of the homologs of glutathione hydrolase in the instant invention are represented by UniProt entries: P18956, P19440, P07314, P54422, Q60928, P20735, P63186, P36269, Q9QWE9, Q9UJ14, Q6P531, Q99JP7, Q99MZ4, Q9Z2A9, P36267, Q9I406, A0A193AUF6, D4B387, Q9LR30, Q9S7E9, O14194, Q9US04, Q0V8L2, Q6PDE7, A0A193AU77, Q6IE08, P74181, and A7YWM1.
  • glutathione hydrolase is provided by the amino acid sequence of SEQ ID NO: 23, which is glutathione hydrolase from Escherichia coli and encoded by the nucleic acid sequence of SEQ ID NO: 24.
  • the UniProt entries listed herein are incorporated by reference in their entireties. ggT (E.
  • NEX-16225 yhaM is a cysteine desulfidase that breaks down cysteine into pyruvate, hydrogen sulfide, and ammonium.
  • An example of desulfidase is provided by the sequence of SEQ ID NO: 25, which is desulfidase from Escherichia coli. Homologues of desulfidase from microorganisms other than Escherichia coli can be used in the microorganisms and methods described herein.
  • Non- limiting examples of the homologs of desulfidase in the instant invention are represented by UniProt entries: P42626, O07521, Q9KDN6, Q5WHU5, A9VI37, A7Z305, Q929F3, Q8Y556, Q5L299, Q63EZ0, Q8CUG1, A0RAN0, C3LCZ8, B9ISF7, Q71XE5, Q6HME9, C1KXH5, C3P2S0, Q65LT6, Q81H06, C5D6L2, A7GLX6, Q81U72, A0AKX3, B7HFZ0, B7IK95, B7HYS8, A8FBK3, Q8XAF6, B7JCU0, C1EKC8, Q73CF4, Q58431, P42628, O34441, B5BGF2, Q8ZLW5, Q5PCB2, B5QZR6, A9MPR6, B5FHX2, Q1R6M3, A9N623, B1
  • desulfidase is provided by the amino acid sequence of SEQ ID NO: 25, which is desulfidase from Escherichia coli and encoded by the nucleic acid sequence of SEQ ID NO: 26.
  • the UniProt entries listed herein are incorporated by reference in their entireties. yhaM (E.
  • yhaO is a L-cysteine importer and is part of a bicistronic operon with YhaM, a L- cysteine desulfidase.
  • Permeases are membrane transport proteins that allow specific molecules to move in or out of cells.
  • An example of L-cysteine importer is provided by the sequence of SEQ ID NO: 27, which is serine permease from Escherichia coli. Homologues of L-cysteine importer from 53 FH12512877.6 Attorney Docket No.: NEX-16225 microorganisms other than Escherichia coli can be used in the microorganisms and methods described herein.
  • Non-limiting examples of the homologs of L-cysteine importers in the instant invention are represented by UniProt entries: P42628, O07522, Q8XAF5, P0ACJ5, P42626, A0A166FB33, A0A223YZZ4, A0A2U1S865, A0A166BYK2, A0A166FBN6, A0A2H5V8N4, A0A2H6JWJ8, A0A2H6GWE9, A0A315XMS1, A0A2A2HC94, A0A1V5P8A9, A0A2H5YWJ3, and A0A2H6AP10.
  • L-cysteine importer is provided by the amino acid sequence of SEQ ID NO: 27, which is L-cysteine importer from Escherichia coli and encoded by the nucleic acid sequence of SEQ ID NO: 28.
  • the UniProt entries listed herein are incorporated by reference in their entireties. yhaO (E.
  • CysI contains one siroheme and one 4-Fe-4S iron-sulfur center per chain. CysI is a gene that encodes the beta chain of the sulfite reductase complex. CysJ is the flavin or alpha subunit of sulfite reductase (SiR), an enzyme that plays a key role in the sulfur cycle.
  • SiR sulfite reductase
  • An example of sulfite reductase (CysI) is provided by the sequence of SEQ ID NO: 29, which is sulfite reductase from Escherichia coli. Homologues of sulfite reductase from microorganisms other than Escherichia coli can be used in the microorganisms and methods described herein.
  • Non-limiting examples of the homologs of sulfite reductase (CysI) in the instant invention are represented by UniProt entries: P17846, P17845, Q9JS33, O32213, Q8VSR0, Q8KQT8, Q6D1A2, Q7VQH1, B5FGI9, Q9KF75, Q5WKE7, P52673, A7MSZ7, A7Z8R5, B7VI85, Q5NRM2, Q66ED3, Q9JUD9, A1KU05, A9IT77, P57502, Q65T54, Q6LM59, Q82W45, Q8P608, B4F234, B5BEZ8, Q2P0H2, Q4L9F1, Q2NVN3, Q5HKZ7, Q7N8L5, Q8PHC8, A0KNL0, A4IMT6, A8A3P4, A8FZY7, B1LQ86, B5FTU2, B8D7V7, C
  • CysI sulfite reductase
  • Homologues of sulfite reductase from microorganisms other than Escherichia coli can be used in the microorganisms and methods described herein.
  • Non-limiting examples of the homologs of sulfite reductase (CysJ) in the instant invention are represented by UniProt entries: P38038, Q9JS45, O32214, P38039, Q8ZBN6, Q6D1A1, Q7VQH2, P52674, Q7N8L6, Q2NVN4, Q5PEH7, Q66ED4, Q65T53, Q31XM4, Q83QD9, Q8EAZ9, Q8K9D3, A4TPY5, A8A3P5, A9MF16, Q8Z458, A0KTH4, A9N2E6, Q8DCK2, A1KU06, P57503, Q5NRM1, Q87L90, Q9JUD8, Q6LM58, Q7MHA5, Q1CLS8, B1IU77,
  • CysJ sulfite reductase
  • SEQ ID NO: 31 is sulfite reductase from Escherichia coli and encoded by the nucleic acid sequence of SEQ ID NO: 32. 57 FH12512877.6 Attorney Docket No.: NEX-16225 The UniProt entries listed herein are incorporated by reference in their entireties.
  • CysG is the enzyme that produces siroheme, an iron-containing porphinoid that is essential for bacterial sulfur metabolism.
  • siroheme synthetase is provided by the sequence of SEQ ID NO: 33, which is siroheme synthetase from Escherichia coli. Homologues of siroheme synthetase from microorganisms other than Escherichia coli can be used in the microorganisms and methods described herein.
  • Non-limiting examples of the homologs of siroheme synthetase in the instant invention are represented by UniProt entries: P0AEA8, Q65T49, Q820Q4, P25924, Q1H3L5, Q2Y6L7, P95370, Q74Y23, Q2KWB0, Q0A812, Q31GG8, P57001, Q606C9, Q6F8G6, Q7WB57, Q7WMM4, A1KU10, A9HZV6, P57500, Q7VQG9, Q9I0M7, B0BTC2, B0U4X0, B4TY38, C0Q0F3, C3JY53, Q32AZ8, Q3YWQ3, A1SRP9, A5CXE4, 59 FH12512877.6 Attorney Docket No.: NEX-16225 A5WEG6, B1X716, B5FJQ1, C4ZUM4, Q1LTP4, Q1R5R3, Q3SG32,
  • siroheme synthetase is provided by the amino acid sequence of SEQ ID NO: 33, which is siroheme synthetase from Escherichia coli and encoded by the nucleic acid sequence of SEQ ID NO: 34.
  • the UniProt entries listed herein are incorporated by reference in their entireties. cysG (E.
  • cysteine desulfurase which catalyzes the transfer of sulfur and selenium from cysteine and selenocysteine to a range of recipients.
  • An example of cysteine desulfurase is provided by the sequence of SEQ ID NO: 35, which is cysteine desulfurase from Escherichia coli. Homologues of cysteine desulfurase from microorganisms other than Escherichia coli can be used in the microorganisms and methods described herein.
  • Non-limiting examples of the homologs of cysteine desulfurase in the instant invention are represented by UniProt entries: P0A6B7, O31269, P0A6B9, O54055, P9WQ71, Q6D259, B0VD51, B5R5A2, Q9HXI8, A7MU48, B7VJS6, B5BAW6, P57795, Q65RS7, Q7MNG2, Q5PNG1, Q60C64, Q7N224, Q87S28, Q8ZN40, B0VNW2, Q68WP6, Q9JTX0, A1KUK1, B4EZU8, P57657, P57803, Q6LU62, Q9JYY0, Q9ZD60, A0KJ32, A1AWM1, A8EYH9, A9N1X5, B8DZS1, C4L7K2, Q1RHY6, Q4UL77, Q8DEY7, Q3IFI3, A0K
  • cysteine desulfurase is provided by the amino acid sequence of SEQ ID NO: 35, which is cysteine desulfurase from Escherichia coli and encoded by the nucleic acid sequence of SEQ ID NO: 36.
  • the UniProt entries listed herein are incorporated by reference in their entireties. 61 FH12512877.6 Attorney Docket No.: NEX-16225 iscS (E.
  • decR is provided by the sequence of SEQ ID NO: 37, which is a DNA-binding transcriptional activator from Escherichia coli. Homologues of decR from microorganisms other than Escherichia coli can be used in the microorganisms and methods described herein.
  • Non-limiting examples of the homologs of decR in the instant invention are represented by UniProt entries: 0ACJ5, P0ACJ6, P0ACJ7, Q22230, Q23116, A0A812T6V2, A0A5E7FH89, A0A3G8JGB0, A0A451D390, A0A451DDE8, A0A517DXY7, A0A5B8R5Y8, A0A7D3P6R8, A0A7X1EFH1, A0A8E6NHW6, A0A8E9YAW5, A0A916I1J9, A0A916MYW8, A0A977N6I8, A0AA35XS47, and A0AAD2V8S9.
  • DNA-binding transcriptional activator is provided by the amino acid sequence of SEQ ID NO: 37, which is DNA-binding transcriptional activator from Escherichia coli and encoded by the nucleic acid sequence of SEQ ID NO: 38.
  • the UniProt entries listed herein are incorporated by reference in their entireties. decR (E.
  • the engineered microorganism may lack activity of these enzymes.
  • activity refers to the total capacity of a cell to perform a function.
  • a genetic modification that decreases the activity of an enzyme in a cell may reduce the amount of the enzyme in a cell or reduce the efficiency of the enzyme.
  • An enzyme knockout reduces the amount of the enzyme in the cell.
  • a mutation to an enzyme gene may reduce the efficiency of its enzyme protein product with little effect on the amount of cellular enzyme.
  • Mutations that reduce the efficiency of the enzyme may affect the active site, for example, by changing one or more active site residues; they may impair the enzyme’s kinetics, for example, by sterically blocking substrates or products; they may affect protein folding or dynamics, for example, by reducing the proportion of properly-folded enzymes; they may affect protein localization; or they may affect protein degradation, for example, by adding one or more protein cleavage sites or by adding one or more residues or amino acid sequences that target the protein for proteolysis. These mutations affect coding regions. Mutations that decrease the enzyme activity may instead affect the transcription or translation of the gene. For example, mutation to an enzyme enhancer or promoter can reduce the enzyme activity by reducing its expression.
  • Mutating or deleting the non-coding portions of an enzyme gene, such as its introns, may also reduce transcription or translation. Additionally, mutations to the upstream regulators of an enzyme may its activity; for example, the over-expression of one or more repressors may decrease the enzyme activity, and a knockout or mutation of one or more activators may similarly decrease the enzyme activity. The enzymes may be knocked out in the engineered microorganism.
  • knockout mutation or “knockout” refers to a genetic modification that prevents a native gene from being transcribed and translated into a functional protein.
  • the term “native” refers to the composition of a cell or parent cell prior to a transformation event.
  • a “native gene” refers to a nucleotide sequence that encodes a protein that has not been introduced into a cell by a transformation event.
  • a “native protein” refers to an amino acid sequence that is encoded by a native gene.
  • the engineered microorganism disclosed herein comprises at least one heterologous gene, which is operably linked to a promoter (e.g., an inducible 64 FH12512877.6 Attorney Docket No.: NEX-16225 promoter) that is activated or repressed by an environmental cue.
  • a promoter e.g., an inducible 64 FH12512877.6 Attorney Docket No.: NEX-16225 promoter
  • the environmental cue is H 2 S, reactive oxygen species (ROS), thiosulfate, or interleukins.
  • the promoter is a ROS-activated promoter such as the oxyR (transcription factor) and oxyS (promoter) gene-promoter from E. coli. The ROS activates the OxyR, which activates the OxyS promoter.
  • OxyR is a transcription factor.
  • An example of OxyR is provided by the sequence of SEQ ID NO: 39, which is OxyR from Escherichia coli.
  • Homologues of OxyR from microorganisms other than Escherichia coli can be used in the microorganisms and methods described herein.
  • Non-limiting examples of the homologs of OxyR in the instant invention are represented by UniProt entries: P0ACQ4, P0ACQ5, P0ACQ6, L8EYU3, P71318, Q9X725, P52677, O87324, P52678, O87883, Q9X5P2, P44418, and Q9HTL4.
  • OxyR is provided by the amino acid sequence of SEQ ID NO: 39, which is OxyR from Escherichia coli and encoded by the nucleic acid sequence of SEQ ID NO: 40.
  • the UniProt entries listed herein are incorporated by reference in their entireties. oxyR (E.
  • OxyS is provided by the nucleic acid sequence of SEQ ID NO: 41, which is OxyS from Escherichia coli. Homologues of OxyS from microorganisms other than Escherichia coli can be used in the microorganisms and methods described herein.
  • OxyS promoter E.
  • the promoter is a luminal H 2 S-activated promoter, such as an IR9 promoter from B. licheniformis.
  • the engineered microorganism expresses NreB and NreC.
  • the NreB senses H 2 S and phosphorylates the NreC, which activates the IR9 promoter from B. licheniformis.
  • nreB senses H 2 S and phosphorylates the NreC.
  • An example of nreB is provided by the sequence of SEQ ID NO: 42, which is nreB from B. licheniformis. Homologues of nreB from microorganisms other than B. licheniformis can be used in the microorganisms and methods described herein.
  • Non-limiting examples of the homologs of nreB in the instant invention are represented by UniProt entries: Q7WZY5, Q4L8Q7, Q5HDG4, Q2YZ41, Q6G6S9, Q5HLK5, Q7A028, Q6GE41, Q931F6, Q99RN7, A6QJN2, A8Z581, Q8CR97, A5IVH3, A6U4C1, Q2FVM6, Q2FEA5, A7X624, O28682, A0A1W7A8K1, A0A1X6ZAH4, and Q5WK46.
  • nreB is provided by the amino acid sequence of SEQ ID NO: 42, which is nreB from B. licheniformis and encoded by the nucleic acid sequence of SEQ ID NO: 43. The UniProt entries listed herein are incorporated by reference in their entireties. nreB (B.
  • nreC is provided by the sequence of SEQ ID NO: 44, which is nreC from B. licheniformis. Homologues of nreC from microorganisms other than B. licheniformis can be used in the microorganisms and methods described herein.
  • nreC is provided by the amino acid sequence of SEQ ID NO: 44, which is nreC from B. licheniformis and encoded by the nucleic acid sequence of SEQ ID NO: 45.
  • the UniProt entries listed herein are incorporated by reference in their entireties. nreC (B.
  • the thiosulfate-activated promoter may be a phsA342 promoter from S. halifaxensis.
  • the engineered microorganism expresses a thiosulfate sensor (e.g., thsS), which activates a transcription factor (e.g., thsR).
  • thsS is a membrane-bound thiosulfate sensor which phosphorylates the transcription factor, thsR, which binds to the phsA342 promoter to drive gene expression from Shewanella halifaxensis.
  • the engineered microorganism expresses a transcription factor that is activated by thiosulfate.
  • the activated transcription factor activates the promoter.
  • thsS is a membrane-bound thiosulfate sensor which phosphorylates the transcription factor, thsR.
  • An example of thsS is provided by the sequence of SEQ ID NO: 47, which is thsS from Shewanella halifaxensis. Homologues of thsS from microorganisms other than Shewanella halifaxensis can be used in the microorganisms and methods described herein.
  • An example of thsS is provided by the amino acid sequence of SEQ ID NO: 47, which is thsS from Shewanella halifaxensis and encoded by the nucleic acid sequence of SEQ ID NO: 48.
  • thsR Homologues of thsR from microorganisms other than Shewanella halifaxensis can be used in the microorganisms and methods described herein.
  • An example of thsR is provided by the amino acid sequence of SEQ ID NO: 49, which is thsR from Shewanella halifaxensis and encoded by the nucleic acid sequence of SEQ ID NO: 50.
  • thsR Shewanella halifaxensis 1 MQQQINGPVY LVDDDEAIID SIDFLMEGYG YKLNSFNCGD RFLAEVDLTQ 51 AGCVILDARM PGLTGPQVQQ LLSDAKSPLA VIFLTGHGDV PMAVDAFKNG 101 AFDFFQKPVP GSLLSQSIAK GLTYSIDQHL KRTNQALIDT LSEREAQIFQ 151 LVIAGNTNKQ MANELCVAIR TIEVHRSKLM TKLGVNNLAE LVKLAPLLAH 201 KSE (SEQ ID NO: 49) 71 FH12512877.6 Attorney Docket No.: NEX-16225 thsR (Shewanella halifaxensis, codon optimized for E.
  • phsA342 is provided by the nucleic acid sequence of SEQ ID NO: 51, which is phsA342 from Shewanella halifaxensis. Homologues of phsA342 from microorganisms other than Shewanella halifaxensis can be used in the microorganisms and methods described herein.
  • the Interleukin may be IL-1, IL-4, IL-5 IL-6, IL-8, IL-10, IL-11, IL-13, IL-17A, IL-17F, IL-27, IL-28, IL-29, IL-36, IL-38, IL-39, or IL-40 and pro- inflammatory may be tumor necrosis factor (TNF), TNF-alpha, TNF-beta, C-reactive protein 72 FH12512877.6 Attorney Docket No.: NEX-16225 (CRP), chemokine ligands (CCL) 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, and 28, and the C-X-C motif chemokine ligand (CXCL) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, XCL1, XCL2, and CX3CL1.
  • the engineered microorganism expresses a transcription factor that is activated by an interleukin or pro-inflammatory cytokine or chemokine.
  • the activated transcription factor activates the promoter.
  • "Inducible promoter” is a promoter that mediates the transcription of an operably linked gene in response to a particular stimulus.
  • D21 Acidilobus saccharovorans, Acidithiobacillus ferrivorans, Acidovorax sp.98_63833, Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter genomosp. C1, Acinetobacter haemolyticus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter lwoffii, Acinetobacter parvus, Acinetobacter radioresistens, Acinetobacter schindleri, Acinetobacter sp.56A1, Acinetobacter sp. CIP 101934, Acinetobacter sp. CIP 102143, Acinetobacter sp.
  • P2P_19 P1 Actinomyces cardiffensis, Actinomyces europaeus, Actinomyces funkei, Actinomyces genomosp. C1, Actinomyces genomosp. C2, Actinomyces genomosp.
  • Actinomyces sp. HKU31 Actinomyces sp. ICM34, Actinomyces sp. ICM41, Actinomyces sp. ICM47, Actinomyces sp. ICM54, Actinomyces sp. M2231_94_1, Actinomyces sp. oral clone GU009, Actinomyces sp. oral clone GU067, Actinomyces sp. oral clone IO076, Actinomyces sp. oral clone IO077, Actinomyces sp. oral clone IP073, Actinomyces sp.
  • TeJ5 Actinomyces urogenitalis, Actinomyces viscosus, Adlercreutzia equolifaciens, Aerococcus sanguinicola, Aerococcus urinae, Aerococcus urinaeequi, Aerococcus viridans, Aeromicrobium marinum, Aeromicrobium sp.
  • RMA 9912 Alkaliphilus metalliredigenes, Alkaliphilus oremlandii, Alloscardovia omnicolens, Alloscardovia sp. OB7196, Anaerobaculum hydrogeniformans, Anaerobiospirillum succiniciproducens, Anaerobiospirillum thomasii, Anaerococcus hydrogenalis, Anaerococcus lactolyticus, Anaerococcus octavius, Anaerococcus prevotii, Anaerococcus sp.8404299, Anaerococcus sp.8405254, Anaerococcus sp.9401487, Anaerococcus sp.9403502, Anaerococcus sp.
  • gpac104 Anaerococcus sp. gpac126, Anaerococcus sp. gpac155, Anaerococcus sp. gpac199, Anaerococcus sp. gpac215, Anaerococcus tetradius, Anaerococcus vaginalis, Anaerofustis stercorihominis, Anaeroglobus geminatus, Anaerosporobacter mobilis, Anaerostipes caccae, Anaerostipes sp.3_2_56FAA, Anaerotruncus colihominis, Anaplasma marginale, Anaplasma phagocytophilum, Aneurinibacillus aneurinilyticus, Aneurinibacillus danicus, Aneurinibacillus migulanus, Aneurinibacillus terranovensis, Aneurinibacillus thermoaerophilus, Anoxybacillus contaminans, Anoxybacillus flavithermus
  • Atopobium sp. F0209 Atopobium sp. ICM42b10, Atopobium sp. ICM57, Atopobium vaginae, Aurantimonas coralicida, Aureimonas altamirensis, Auritibacter ignavus, Averyella dalhousiensis, Bacillus aeolius, Bacillus aerophilus, Bacillus aestuarii, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus anthracis, Bacillus atrophaeus, Bacillus badius, Bacillus cereus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus flexus, Bacillus fordii, Bacillus gelatini, Bacillus halmapalus, Bacillus halodurans, Bacillus 75 FH12512877.6 Attorney Docket No.:
  • P8 oral clone MB4_G15 Bacteroides acidifaciens, Bacteroides barnesiae, Bacteroides caccae, Bacteroides cellulosilyticus, Bacteroides clarus, Bacteroides coagulans, Bacteroides coprocola, Bacteroides coprophilus, Bacteroides dorei, Bacteroides eggerthii, Bacteroides faecis, Bacteroides finegoldii, Bacteroides fluxus, Bacteroides fragilis, Bacteroides galacturonicus, Bacteroides helcogenes, Bacteroides heparinolyticus, Bacteroides intestinalis, Bacteroides massiliensis, Bacteroides nordii, Bacteroides oleiciplenus, Bacteroides ovatus, Bacteroides pectinophilus, Bacteroides plebeius, Bacteroides pyogenes, Bacteroides salanitronis
  • XB12B Bacteroides sp. XB44A, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides ureolyticus, Bacteroides vulgatus, 76 FH12512877.6 Attorney Docket No.: NEX-16225 Bacteroides xylanisolvens, Bacteroidetes bacterium oral taxon D27, Bacteroidetes bacterium oral taxon F31, Bacteroidetes bacterium oral taxon F44, Barnesiella intestinihominis, Barnesiella viscericola, Bartonella bacilliformis, Bartonella grahamii, Bartonella henselae, Bartonella quintana, Bartonella tamiae, Bartonella washoensis, Bdellovibrio sp.
  • MPA Bifidobacteriaceae genomosp. C1, Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium gallicum, Bifidobacterium infantis, Bifidobacterium kashiwanohense, Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium pseudolongum, Bifidobacterium scardovii, Bifidobacterium sp.
  • HM2 Bifidobacterium sp. HMLN12, Bifidobacterium sp. M45, Bifidobacterium sp. MSX5B, Bifidobacterium sp. TM_7, Bifidobacterium thermophilum, Bifidobacterium urinalis, Bilophila wadsworthia, Bisgaard Taxon, Bisgaard Taxon, Bisgaard Taxon, Bisgaard Taxon, Blastomonas natatoria, Blautia coccoides, Blautia glucerasea, Blautia glucerasei, Blautia hansenii, Blautia hydrogenotrophica, Blautia luti, Blautia producta, Blautia schinkii, Blautia sp.
  • HIS5 Brevibacillus agri, Brevibacillus brevis, Brevibacillus centrosporus, Brevibacillus choshinensis, Brevibacillus invocatus, Brevibacillus laterosporus, Brevibacillus parabrevis, Brevibacillus reuszeri, Brevibacillus sp. phR, Brevibacillus thermoruber, Brevibacterium aurantiacum, Brevibacterium casei, Brevibacterium epidermidis, Brevibacterium frigoritolerans, Brevibacterium linens, Brevibacterium mcbrellneri, Brevibacterium paucivorans, Brevibacterium sanguinis, Brevibacterium sp.
  • YIT 12070 Clostridium sphenoides, Clostridium spiroforme, Clostridium sporogenes, Clostridium sporosphaeroides, Clostridium stercorarium, Clostridium sticklandii, Clostridium straminisolvens, Clostridium subterminale, Clostridium sulfidigenes, Clostridium symbiosum, Clostridium tertium, Clostridium tetani, Clostridium thermocellum, Clostridium tyrobutyricum, Clostridium viride, Clostridium xylanolyticum, Collinsella aerofaciens, Collinsella intestinalis, Collinsella stercoris, Collinsella tanakaei, Comamonadaceae bacterium NML000135, Comamonadaceae bacterium NML790751, Comamonadaceae bacterium NML910035, Comamonadaceae
  • NSP5 Conchiformibius kuhniae, Coprobacillus cateniformis, Coprobacillus sp.29_1, Coprobacillus sp. D7, Coprococcus catus, Coprococcus comes, Coprococcus eutactus, Coprococcus sp.
  • Coriobacteriaceae bacterium JC110 Coriobacteriaceae bacterium phI, Corynebacterium accolens, Corynebacterium ammoniagenes, Corynebacterium amycolatum, Corynebacterium appendicis, Corynebacterium argentoratense, Corynebacterium atypicum, Corynebacterium aurimucosum, Corynebacterium bovis, Corynebacterium canis, Corynebacterium casei, Corynebacterium confusum, Corynebacterium coyleae, Corynebacterium diphtheriae, Corynebacterium durum, Corynebacterium efficiens, Corynebacterium falsenii, Corynebacterium flav
  • oral clone JV001 Deferribacteres sp. oral clone JV006, Deferribacteres sp. oral clone JV023, Deinococcus radiodurans, Deinococcus sp. R_43890, Delftia acidovorans, Dermabacter hominis, Dermacoccus sp. Ellin185, Desmospora activa, Desmospora sp.8437, Desulfitobacterium frappieri, Desulfitobacterium hafniense, Desulfobulbus sp.
  • TSE38 Enterobacteriaceae bacterium 9_2_54FAA, Enterobacteriaceae bacterium CF01Ent_1, Enterobacteriaceae bacterium Smarlab 3302238, Enterococcus avium, Enterococcus caccae, Enterococcus casseliflavus, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus gilvus, Enterococcus hawaiiensis, Enterococcus hirae, Enterococcus italicus, Enterococcus mundtii, Enterococcus raffinosus, Enterococcus sp.
  • BV2CASA2 Enterococcus sp. CCRI_16620, Enterococcus sp. F95, Enterococcus sp. RfL6, Enterococcus thailandicus, Eremococcus coleocola, Erysipelothrix inopinata, Erysipelothrix rhusiopathiae, Erysipelothrix tonsillarum, Erysipelotrichaceae bacterium 3_1_53, Erysipelotrichaceae bacterium 5_2_54FAA, Escherichia albertii, Escherichia coli, Escherichia fergusonii, Escherichia hermannii, Escherichia sp.1_1_43, Escherichia sp.
  • WAL 14571 Eubacterium ska, Eubacterium tortuosum, Eubacterium ventriosum, Eubacterium xylanophilum, Eubacterium yurii, Ewingella americana, Exiguobacterium acetylicum, Facklamia hominis, Faecalibacterium prausnitzii, Filifactor alocis, Filifactor villosus, Finegoldia magna, Flavobacteriaceae genomosp. C1, Flavobacterium sp.
  • NF2_1 Flavonifractor plautii, Flexispira rappini, Flexistipes sinusarabici, Francisella novicida, Francisella philomiragia, Francisella tularensis, Fulvimonas sp. NML 060897, 81 FH12512877.6
  • Fusobacterium gonidiaformans Fusobacterium mortiferum, Fusobacterium naviforme, Fusobacterium necrogenes, Fusobacterium necrophorum, Fusobacterium nucleatum, Fusobacterium periodonticum, Fusobacterium russii, Fusobacterium sp.1_1_41FAA, Fusobacterium sp.11_3_2, Fusobacterium sp.12_1B, Fusobacterium sp.2_1_31, Fusobacterium sp.3_1_27, Fusobacterium sp.3_1_33, Fusobacterium sp.3_1_36A2, Fusobacterium sp.3_1_5R, Fusobacterium sp.
  • oral clone ASCG05 Grimontia hollisae, Haematobacter sp. BC14248, Haemophilus aegyptius, Haemophilus ducreyi, Haemophilus genomosp.
  • P2 oral clone MB3_C24 Haemophilus genomosp.
  • P3 oral clone MB3_C38 Haemophilus haemolyticus, Haemophilus influenzae, Haemophilus parahaemolyticus, Haemophilus parainfluenzae, Haemophilus paraphrophaemolyticus, Haemophilus parasuis, Haemophilus somnus, Haemophilus sp. 70334, Haemophilus sp. HK445, Haemophilus sp. oral clone ASCA07, Haemophilus sp. oral clone ASCG06, Haemophilus sp. oral clone BJ021, Haemophilus sp.
  • SRC_DSD1 Klebsiella sp. SRC_DSD11, Klebsiella sp. SRC_DSD12, Klebsiella sp. SRC_DSD15, Klebsiella sp. SRC_DSD2, Klebsiella sp.
  • Lactobacillus acidipiscis Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus antri, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus catenaformis, Lactobacillus coleohominis, Lactobacillus coryniformis, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus dextrinicus, Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus gastricus, Lactobacillus genomosp.
  • CCUG 43427A Megamonas funiformis, Megamonas hypermegale, Megasphaera elsdenii, Megasphaera genomosp. C1, Megasphaera genomosp. type_1, Megasphaera micronuciformis, Megasphaera sp. BLPYG_07, Megasphaera sp.
  • MM4 Methylocella silvestris, Methylophilus sp. ECd5, Microbacterium chocolatum, Microbacterium flavescens, Microbacterium gubbeenense, Microbacterium lacticum, Microbacterium oleivorans, Microbacterium oxydans, Microbacterium paraoxydans, Microbacterium phyllosphaerae, Microbacterium schleiferi, Microbacterium sp.768, Microbacterium sp.
  • JB_T16 Morococcus cerebrosus, Moryella indoligenes, Mycobacterium abscessus, Mycobacterium africanum, Mycobacterium alsiensis, Mycobacterium avium, Mycobacterium chelonae, Mycobacterium colombiense, Mycobacterium elephantis, Mycobacterium gordonae, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium lacus, Mycobacterium leprae, Mycobacterium lepromatosis, Mycobacterium mageritense, Mycobacterium mantenii, Mycobacterium marinum, Mycobacterium microti, Mycobacterium neoaurum, Mycobacterium parascrofulaceum, Mycobacterium paraterrae, Mycobacterium phlei, Mycobacterium seoulense, Mycobacterium smegmatis, Mycobacterium sp.1761, Mycobacterium sp.1776, Mycobacter
  • Mycobacterium sp. B10_07.09.0206 Mycobacterium sp. GN_10546, Mycobacterium sp. GN_10827, Mycobacterium sp. GN_11124, Mycobacterium sp. GN_9188, Mycobacterium sp. GR_2007_210, Mycobacterium sp. HE5, Mycobacterium sp. NLA001000736, Mycobacterium sp.
  • W Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycobacterium vulneris, Mycoplasma agalactiae, Mycoplasma amphoriforme, Mycoplasma arthritidis, Mycoplasma bovoculi, Mycoplasma faucium, Mycoplasma fermentans, Mycoplasma flocculare, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma orale, Mycoplasma ovipneumoniae, Mycoplasma penetrans, Mycoplasma pneumoniae, Mycoplasma putrefaciens, Mycoplasma salivarium, 85 FH12512877.6 Attorney Docket No.: NEX-16225 Mycoplasmataceae genomosp.
  • P1 oral clone MB1_G23 Myroides odoratimimus, Myroides sp. MY15, Neisseria bacilliformis, Neisseria cinerea, Neisseria elongata, Neisseria flavescens, Neisseria genomosp.
  • P2 oral clone MB5_P15 Neisseria gonorrhoeae, Neisseria lactamica, Neisseria macacae, Neisseria meningitidis, Neisseria mucosa, Neisseria pharyngis, Neisseria polysaccharea, Neisseria sicca, Neisseria sp.
  • Neisseria sp. oral clone AP132 Neisseria sp. oral clone JC012
  • Neisseria sp. oral strain B33KA Neisseria sp. oral taxon 014
  • Neisseria sp. SMC_A9199 Neisseria sp.
  • TM10_1 Neisseria subflava, Neorickettsia risticii, Neorickettsia sennetsu, Nocardia brasiliensis, Nocardia cyriacigeorgica, Nocardia farcinica, Nocardia puris, Nocardia sp.01_Je_025, Nocardiopsis rougevillei, Novosphingobium aromaticivorans, Oceanobacillus caeni, Oceanobacillus sp.
  • Ndiop Ochrobactrum anthropi, Ochrobactrum intermedium, Ochrobactrum pseudintermedium, Odoribacter laneus, Odoribacter splanchnicus, Okadaella gastrococcus, Oligella ureolytica, Oligella urethralis, Olsenella genomosp. C1, Olsenella profusa, Olsenella sp. F0004, Olsenella sp. oral taxon 809, Olsenella uli, Opitutus terrae, Oribacterium sinus, Oribacterium sp. ACB1, Oribacterium sp. ACB7, Oribacterium sp.
  • G2 Oscillibacter valericigenes, Oscillospira guilliermondii, Oxalobacter formigenes, Paenibacillus barcinonensis, Paenibacillus barengoltzii, Paenibacillus chibensis, Paenibacillus cookii, Paenibacillus durus, Paenibacillus glucanolyticus, Paenibacillus lactis, Paenibacillus lautus, Paenibacillus pabuli, Paenibacillus polymyxa, Paenibacillus popilliae, Paenibacillus sp.
  • CIP 101062 Paenibacillus sp.
  • HGF5 Paenibacillus sp. HGF7, Paenibacillus sp. JC66, Paenibacillus sp. oral taxon F45, Paenibacillus sp. R_27413, Paenibacillus sp.
  • R_27422 Paenibacillus timonensis, Pantoea agglomerans, Pantoea ananatis, Pantoea brenneri, Pantoea citrea, Pantoea conspicua, Pantoea septica, Papillibacter cinnamivorans, Parabacteroides distasonis, Parabacteroides goldsteinii, Parabacteroides gordonii, Parabacteroides johnsonii, Parabacteroides merdae, Parabacteroides sp. D13, Parabacteroides sp. NS31_3, Parachlamydia sp.
  • oral taxon 836 Peptostreptococcaceae bacterium ph1, Peptostreptococcus anaerobius, Peptostreptococcus micros, Peptostreptococcus sp.9succ1, Peptostreptococcus sp. oral clone AP24, Peptostreptococcus sp. oral clone FJ023, Peptostreptococcus sp. P4P_31 P3, Peptostreptococcus stomatis, Phascolarctobacterium faecium, Phascolarctobacterium sp.
  • YIT 12068 Phascolarctobacterium succinatutens, Phenylobacterium zucineum, Photorhabdus asymbiotica, Pigmentiphaga daeguensis, Planomicrobium koreense, Plesiomonas shigelloides, Porphyromonadaceae bacterium NML 060648, Porphyromonas asaccharolytica, Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonas levii, Porphyromonas macacae, Porphyromonas somerae, Porphyromonas sp. oral clone BB134, Porphyromonas sp.
  • C1 Prevotella genomosp.
  • C2 Prevotella genomosp. P7 oral clone MB2_P31, Prevotella genomosp. P8 oral clone MB3_P13, Prevotella genomosp.
  • P9 oral clone MB7_G16 Prevotella heparinolytica, Prevotella histicola, Prevotella intermedia, Prevotella loescheii, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella ruminicola, Prevotella salivae, Prevotella sp.
  • Prevotella sp. CM38 Prevotella sp. ICM1, Prevotella sp. ICM55, Prevotella sp. JCM 6330, Prevotella sp. oral clone AA020, Prevotella sp. oral clone ASCG10, Prevotella sp. oral clone ASCG12, Prevotella sp. oral clone AU069, Prevotella sp. oral clone CY006, Prevotella sp. oral clone DA058, Prevotella sp. oral clone FL019, Prevotella sp.
  • Prevotella stercorea Prevotella tannerae, Prevotella timonensis, Prevotella veroralis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella fusca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopos, Prevotella shahii, Prevotella zoogleoformans, Prevotellaceae bacter
  • Propionibacterium sp. LG Propionibacterium sp. oral taxon 192, Propionibacterium sp. S555a, Propionibacterium thoenii, Proteus mirabilis, Proteus penneri, Proteus sp. HS7514, Proteus vulgaris, Providencia alcalifaciens, Providencia rettgeri, Providencia rustigianii, Providencia stuartii, Pseudoclavibacter sp.
  • Timone Pseudoflavonifractor capillosus, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas gessardii, Pseudomonas mendocina, Pseudomonas monteilii, Pseudomonas poae, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas sp.2_1_26, Pseudomonas sp. G1229, Pseudomonas sp.
  • NP522b Pseudomonas stutzeri, Pseudomonas tolaasii, Pseudomonas viridiflava, Pseudoramibacter alactolyticus, Psychrobacter arcticus, Psychrobacter cibarius, Psychrobacter cryohalolentis, Psychrobacter faecalis, Psychrobacter nivimaris, Psychrobacter pulmonis, Psychrobacter sp.13983, Pyramidobacter piscolens, Ralstonia pickettii, Ralstonia sp.5_7_47FAA, Raoultella ornithinolytica, Raoultella planticola, Raoultella terrigena, Rhodobacter sp.
  • Rhodobacter sphaeroides Rhodococcus corynebacterioides, Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus fascians, Rhodopseudomonas palustris, Rickettsia akari, Rickettsia conorii, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia slovaca, Rickettsia typhi, Robinsoniella peoriensis, Roseburia stiicola, Roseburia faecalis, Roseburia faecis, Roseburia hominis, Roseburia intestinalis, Roseburia inulinivorans, Roseburia sp.11SE37, Roseburia sp.
  • Staphylococcus sp. H292 Staphylococcus sp. H780, Staphylococcus succinus, Staphylococcus vitulinus, Staphylococcus warneri, Staphylococcus xylosus, Stenotrophomonas maltophilia, Stenotrophomonas sp.
  • Streptobacillus moniliformis Streptococcus agalactiae, Streptococcus alactolyticus, Streptococcus anginosus, Streptococcus australis, Streptococcus bovis, Streptococcus canis, Streptococcus constellatus, Streptococcus cristatus, Streptococcus downei, Streptococcus dysgalactiae, Streptococcus equi, Streptococcus equinus, Streptococcus gallolyticus, Streptococcus genomosp.
  • Streptococcus genomosp. C3 Streptococcus genomosp. C4, Streptococcus genomosp. C5, Streptococcus genomosp. C6, Streptococcus genomosp. C7, Streptococcus genomosp.
  • Streptococcus gordonii Streptococcus infantarius, Streptococcus infantis, Streptococcus intermedius, Streptococcus lutetiensis, Streptococcus massiliensis, Streptococcus milleri, Streptococcus mitis, Streptococcus mutans, Streptococcus oligofermentans, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus pasteurianus, Streptococcus peroris, Streptococcus pneumoniae, Streptococcus porcinus, Streptococcus pseudopneumoniae, Streptococcus pseudoporcinus, Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sinensis, Streptococcus sp
  • Streptococcus sp. ACS2 Streptococcus sp. AS20, Streptococcus sp. BS35a, Streptococcus sp. C150, Streptococcus sp. CM6, Streptococcus sp. CM7, Streptococcus sp. ICM10, Streptococcus sp. ICM12, Streptococcus sp. ICM2, Streptococcus sp. ICM4, Streptococcus sp. ICM45, Streptococcus sp. M143, Streptococcus sp. M334, Streptococcus sp.
  • Streptococcus suis Streptococcus thermophilus
  • Streptococcus uberis Streptococcus urinalis
  • Streptococcus vestibularis Streptococcus viridans
  • Streptomyces albus Streptomyces griseus
  • Streptomyces sp.1 AIP_2009 Streptomyces sp. SD 511, Streptomyces sp. SD 524, Streptomyces sp. SD 528, Streptomyces sp.
  • SD 534 Streptomyces thermoviolaceus, Subdoligranulum variabile, Succinatimonas hippei, Sutterella morbirenis, Sutterella parvirubra, Sutterella sanguinus, Sutterella sp. YIT 12072, Sutterella stercoricanis, Sutterella wadsworthensis, Synergistes genomosp. C1, Synergistes sp.
  • oral clone ASCG02 Veillonella sp. oral clone OH1A, Veillonella sp. oral taxon 158, Veillonellaceae bacterium oral taxon 131, Veillonellaceae bacterium oral taxon 155, Vibrio cholerae, Vibrio fluvialis, Vibrio furnissii, Vibrio mimicus, Vibrio parahaemolyticus, Vibrio sp.
  • RC341 Vibrio vulnificus, Victivallaceae bacterium NML 080035, Victivallis vadensis, Virgibacillus proomii, Weissella beninensis, Weissella cibaria, Weissella confusa, Weissella hellenica, Weissella kandleri, Weissella koreensis, Weissella paramesenteroides, Weissella sp.
  • KLDS 7.0701 Wolinella succinogenes, Xanthomonadaceae bacterium NML 03_0222, Xanthomonas campestris, Xanthomonas sp.
  • the bacterium is selected from the group consisting of Bacillus, Bacteroides, Bifidobacterium, Clostridium, Corynebacterium, Escherichia, Lactobacillus, Lactococcus, Pseudomonas, Streptomyces, or Mycobacterium.
  • the bacterium is Escherichia coli strain Nissle, MG1655, or S1030.
  • the bacterium is an auxotroph in a gene that is important for cellular growth. Auxotrophy is the inability of an organism to synthesize a particular organic compound required for its growth (as defined by IUPAC) (Iwasaki T., et al.
  • auxotroph is an organism that displays this characteristic. Auxotrophy is the opposite of prototrophy, which is characterized by the ability to synthesize all the compounds needed for growth. 92 FH12512877.6 Attorney Docket No.: NEX-16225 As used herein, the term “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.
  • 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 a DNA 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.
  • 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 gene that is important for cellular growth is dapA, xylA, thyA, tyrA, glnA, cysE, metA, thrC, aspC, hisG, tyrB, proC, lysA, asnA, asnB, or any combination thereof.
  • the bacterium comprises a kill switch.
  • the kill switch is intended to actively kill engineered microbes in response to external stimuli. As opposed to an auxotrophic mutation where bacteria die because they lack an essential nutrient for survival, the kill switch is triggered by a particular factor in the environment that induces the production of toxic molecules within the microbe that cause cell death.
  • Bacteria engineered with kill switches have been engineered for in vitro research purposes, e.g., to limit the spread of a biofuel-producing microorganism outside of a laboratory environment.
  • Bacteria engineered for in vivo administration to treat a disease or disorder may also be programmed to die at a specific time after the expression and delivery of a heterologous gene or genes, for example, a therapeutic gene(s) or after the subject has experienced the therapeutic effect.
  • the kill switch is activated to kill the bacteria after a period of time following oxygen level-dependent expression of arg Afbr .
  • the kill switch is activated in a delayed fashion following oxygen level-dependent expression of arg Afbr , for example, after the production of arginine or citrulline.
  • the bacteria may be engineered to die after the bacteria has spread outside of a disease site.
  • toxins that can be used in kill-switches include, but are not limited to, bacteriocins, lysins, and other molecules that cause cell death by lysing cell membranes, degrading cellular DNA, or other mechanisms. Such toxins can be used individually or in combination.
  • the switches that control their production can be based on, for example, transcriptional activation (toggle switches; see, e.g., Gardner et al., 2000), translation (riboregulators), or DNA recombination (recombinase-based switches), and can sense environmental stimuli such as anaerobiosis or reactive oxygen species. These switches can be activated by a single environmental factor or may require several activators in AND, OR, NAND and NOR logic configurations to induce cell death.
  • transcriptional activation toggle switches; see, e.g., Gardner et al., 2000
  • translation riboregulators
  • DNA recombination recombination-based switches
  • an AND riboregulator switch is activated by tetracycline, isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG), and arabinose to induce the expression of lysins, which permeabilize the cell 94 FH12512877.6 Attorney Docket No.: NEX-16225 membrane and kill the cell. IPTG induces the expression of the endolysin and holin mRNAs, which are then derepressed by the addition of arabinose and tetracycline. All three inducers must be present to cause cell death. Examples of kill switches are known in the art (Callura et al., 2010).
  • the kill switch is activated to kill the bacteria after a period of time following oxygen level-dependent expression of arg Afbr . In some embodiments, the kill switch is activated in a delayed fashion following oxygen level-dependent expression of arg Afbr .
  • Kill-switches can be designed such that a toxin is produced in response to an environmental condition or external signal (e.g., the bacteria is killed in response to an external cue) or, alternatively designed such that a toxin is produced once an environmental condition no longer exists or an external signal is ceased.
  • the bacterial formulation comprises a bacterium and/or a combination of bacteria described herein and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier at least 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%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 7
  • the bacterial composition comprises at least 1 x 10 3 colony forming units (CFUs), 1 x 10 4 colony forming units (CFUs), 1 x 10 5 colony forming units (CFUs), 5 x 10 5 colony forming units (CFUs), 1 x 10 6 colony forming units (CFUs), 2 x 10 6 colony forming units (CFUs), 3 x 10 6 colony forming units (CFUs), 4 x 10 6 colony forming units (CFUs), 5 x 10 6 colony forming units (CFUs), 6 x 10 6 colony forming units (CFUs), 7 x 10 6 colony forming units (CFUs), 8 x 10 6 colony forming units (CFUs), 9 x 10 6 colony forming units (CFUs), 1 x 10 7 colony forming units (CFUs), 2 x 10 7 colony forming units (CFUs)
  • the selected dosage level will depend upon a variety of factors including the subject’s diet, the route of administration, the time of administration, the residence time of the particular microorganism being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could prescribe and/or administer doses of the bacteria employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • probiotic formulations containing the bacteria described herein are provided as encapsulated, enteric coated, or powder forms, with doses ranging up to 10 11 cfu (e.g., up to 10 10 cfu).
  • the composition comprises 5 x 10 11 cfu of the bacteria described herein and 10% (w/w) corn starch in a capsule.
  • the capsule is enteric coated, e.g., for duodenal release at pH 5.5.
  • the composition comprises a powder of freeze-dried of the bacteria described herein which is deemed to have “Qualified Presumption of Safety” (QPS) status.
  • QPS Quality of Safety
  • the composition is storage-stable at frozen or refrigerated temperature.
  • “stably stored” or “storage-stable” refer to a composition in which cells are able to withstand storage for extended periods of time (e.g., at least one month, or two, three, four, six, or twelve months or more) with a less than 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 1% decrease in cell viability.
  • 96 FH12512877.6 Attorney Docket No.: NEX-16225
  • the agar or broth may contain nutrients that provide essential elements and specific factors that enable growth.
  • An example would be a medium composed of 20 g/L glucose, 10 g/L yeast extract, 10 g/L soy peptone, 2 g/L citric acid, 1.5 g/L sodium phosphate monobasic, 100 mg/L ferric ammonium citrate, 80 mg/L magnesium sulfate, 10 mg/L hemin chloride, 2 mg/L calcium chloride, 1 mg/L menadione.
  • Another example would be a medium composed of 10 g/L beef extract, 10 g/L peptone, 5 g/L sodium chloride, 5 g/L dextrose, 3 g/L yeast extract, 3 g/L sodium acetate, 1 g/L soluble starch, and 0.5 g/L L-cysteine HCl, at pH 6.8.
  • a variety of microbiological media and variations are well known in the art (e.g., R.M. Atlas, Handbook of Microbiological Media (2010) CRC Press). Culture media can be added to the culture at the start, may be added during the culture, or may be intermittently/continuously flowed through the culture.
  • the strains in the bacterial composition may be cultivated alone, as a subset of the microbial composition, or as an entire collection comprising the microbial composition.
  • a first strain may be cultivated together with a second strain in a mixed continuous culture, at a dilution rate lower than the maximum growth rate of either cell to prevent the culture from washing out of the cultivation.
  • the inoculated culture is incubated under favorable conditions for a time sufficient to build biomass. For microbial compositions for human use this is often at 37°C temperature, pH, and other parameter with values similar to the normal human niche.
  • the environment may be actively controlled, passively controlled (e.g., via buffers), or allowed to drift.
  • an anoxic/reducing environment may be employed for anaerobic bacterial compositions. This can be accomplished by addition of reducing agents such as cysteine to the broth, and/or stripping it of oxygen.
  • reducing agents such as cysteine
  • a culture of a bacterial composition may be grown at 37°C, pH 7, in the medium above, pre-reduced with 1 g/L cysteine-HCl. When the culture has generated sufficient biomass, it may be preserved for banking.
  • the organisms may be placed into a chemical milieu that protects from freezing (adding ‘cryoprotectants’), drying (‘lyoprotectants’), and/or osmotic shock (‘osmoprotectants’), dispensing into multiple (optionally identical) containers to create a uniform bank, and then treating the culture for preservation.
  • Containers are generally impermeable and have closures that assure isolation from the environment.
  • Cryopreservation treatment is accomplished by freezing a liquid at ultra-low temperatures (e.g., at or below -80°C).
  • Dried preservation removes water from the culture by evaporation (in the case of spray drying or ‘cool drying’) or by sublimation (e.g., for freeze drying, spray freeze drying).
  • Microbial composition banking may be done by culturing and preserving the strains individually, or by mixing the strains together to create a combined bank.
  • a microbial composition culture may be harvested by centrifugation to pellet the cells from the culture medium, the supernatant decanted and replaced with fresh culture broth containing 15% glycerol. The culture can then be aliquoted into 1 mL cryotubes, sealed, and placed at -80°C for long-term viability retention. This procedure achieves acceptable viability upon recovery from frozen storage.
  • Microbial production may be conducted using similar culture steps to banking, including medium composition and culture conditions described above. It may be conducted at larger scales of operation, especially for clinical development or commercial production. At larger scales, there may be several subcultivations of the microbial composition prior to the final cultivation. At the end of cultivation, the culture is harvested to enable further formulation into a dosage form for administration. This can involve concentration, removal of undesirable medium components, and/or introduction into a chemical milieu that preserves the microbial composition and renders it acceptable for administration via the chosen route.
  • a microbial composition may be cultivated to a concentration of 10 10 CFU/mL, then concentrated 20-fold by tangential flow microfiltration; the spent medium may be exchanged by diafiltering with a preservative medium consisting of 2% gelatin, 100 mM trehalose, and 10 mM sodium phosphate buffer.
  • the suspension can then be freeze-dried to a powder and titrated. After drying, the powder may be blended to an appropriate potency, and mixed with other cultures and/or a filler such as microcrystalline cellulose for consistency and ease of handling, and the bacterial composition formulated as provided herein.
  • the bacterial compositions are combined with additional active and/or inactive materials in order to produce a final product, which may be in single dosage unit or in a multi-dose format.
  • the composition comprises at least one carbohydrate.
  • a “carbohydrate” refers to a sugar or polymer of sugars.
  • saccharide polysaccharide
  • carbohydrate and “oligosaccharide” may be used interchangeably.
  • Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom of the molecule. Carbohydrates generally have the molecular formula C n H 2n O n .
  • a carbohydrate may be a monosaccharide, a disaccharide, trisaccharide, oligosaccharide, or polysaccharide.
  • the most basic carbohydrate is a monosaccharide, such as glucose, sucrose, 98 FH12512877.6 Attorney Docket No.: NEX-16225 galactose, mannose, ribose, arabinose, xylose, and fructose.
  • Disaccharides are two joined monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose.
  • an oligosaccharide includes between three and six monosaccharide units (e.g., raffinose, stachyose), and polysaccharides include six or more monosaccharide units.
  • Exemplary polysaccharides include starch, glycogen, and cellulose.
  • Carbohydrates may contain modified saccharide units such as 2’-deoxyribose wherein a hydroxyl group is removed, 2’-fluororibose wherein a hydroxyl group is replaced with a fluorine, or N- acetylglucosamine, a nitrogen-containing form of glucose (e.g., 2’-fluororibose, deoxyribose, and hexose).
  • Carbohydrates may exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.
  • the composition comprises at least one lipid.
  • a “lipid” includes fats, oils, triglycerides, cholesterol, phospholipids, fatty acids in any form including free fatty acids. Fats, oils and fatty acids can be saturated, unsaturated (cis or trans) or partially unsaturated (cis or trans).
  • the lipid comprises at least one fatty acid selected from lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20:1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoic acid (22:1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6) (DHA), and t
  • the composition comprises at least one modified lipid, for example a lipid that has been modified by cooking.
  • the composition comprises at least one supplemental mineral or mineral source.
  • minerals include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium.
  • Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.
  • the composition comprises at least one supplemental vitamin.
  • the at least one vitamin can be fat-soluble or water-soluble vitamins.
  • Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin B12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin.
  • Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the 99 FH12512877.6 Attorney Docket No.: NEX-16225 vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.
  • the composition comprises an excipient.
  • Non-limiting examples of suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.
  • the excipient is a buffering agent.
  • suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.
  • the excipient comprises a preservative.
  • Non-limiting examples of suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.
  • the composition comprises a binder as an excipient.
  • suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.
  • the composition comprises a lubricant as an excipient.
  • suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.
  • the composition comprises a dispersion enhancer as an excipient.
  • compositions of the present invention are combined with a carrier (e.g., a pharmaceutically acceptable carrier) which is physiologically compatible with the gastrointestinal tissue of the subject(s) to which it is administered.
  • a carrier e.g., a pharmaceutically acceptable carrier
  • Carriers can be comprised of solid-based, dry materials for formulation into tablet, capsule or powdered form; or the carrier can be comprised of liquid or gel-based materials for formulations into liquid or gel forms.
  • the specific type of carrier, as well as the final formulation depends, in 100 FH12512877.6 Attorney Docket No.: NEX-16225 part, upon the selected route(s) of administration.
  • the therapeutic composition of the present invention may also include a variety of carriers and/or binders.
  • the carrier is micro-crystalline cellulose (MCC) added in an amount sufficient to complete the one gram dosage total weight.
  • Carriers can be solid-based dry materials for formulations in tablet, capsule or powdered form, and can be liquid or gel-based materials for formulations in liquid or gel forms, which forms depend, in part, upon the routes of administration.
  • Typical carriers for dry formulations include, but are not limited to: trehalose, malto-dextrin, rice flour, microcrystalline cellulose (MCC) magnesium sterate, inositol, FOS, GOS, dextrose, sucrose, and like carriers.
  • Suitable liquid or gel-based carriers include but are not limited to: water and physiological salt solutions; urea; alcohols and derivatives (e.g., methanol, ethanol, propanol, butanol); glycols (e.g., ethylene glycol, propylene glycol, and the like).
  • water-based carriers possess a neutral pH value (i.e., pH 7.0).
  • the composition comprises a disintegrant as an excipient.
  • the disintegrant is a non-effervescent disintegrant.
  • suitable non-effervescent disintegrants include starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pectin, and tragacanth.
  • the composition is a food product (e.g., a food or beverage) such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
  • a food product e.g., a food or beverage
  • a food or beverage such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
  • the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products, including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like.
  • beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages
  • the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, capsules, liquids, pastes, and jellies.
  • the composition may be a fermented food product, such as, but not limited to, a fermented milk product.
  • fermented food products include kombucha, sauerkraut, pickles, miso, tempeh, natto, kimchi, raw cheese, and yogurt.
  • the composition may also be a food additive, such as, but not limited to, an acidulent (e.g., vinegar). Food additives can be divided into several groups based on their effects.
  • Non-limiting examples of food additives include acidulents (e.g., vinegar, citric acid, tartaric acid, malic acid, fumaric acid, and lactic acid), acidity regulators, anticaking agents, antifoaming agents, foaming agents, antioxidants (e.g., vitamin C), bulking agents (e.g., starch), food coloring, fortifying agents, color retention agents, emulsifiers, flavors and flavor enhancers (e.g., monosodium glutamate), flour treatment agents, glazing agents, humectants, tracer gas, preservatives, stabilizers, sweeteners, and thickeners.
  • the bacteria disclosed herein are administered in conjunction with a prebiotic to the subject.
  • Prebiotics are carbohydrates which are generally indigestible by a host animal and are selectively fermented or metabolized by bacteria.
  • Prebiotics may be short-chain carbohydrates (e.g., oligosaccharides) and/or simple sugars (e.g., mono- and di- saccharides) and/or mucins (heavily glycosylated proteins) that alter the composition or metabolism of a microbiome in the host.
  • the short chain carbohydrates are also referred to as oligosaccharides, and usually contain from 2 or 3 and up to 8, 9, 10, 15 or more sugar 102 FH12512877.6 Attorney Docket No.: NEX-16225 moieties.
  • a prebiotic composition can selectively stimulate the growth and/or activity of one of a limited number of bacteria in a host.
  • Prebiotics include oligosaccharides such as fructooligosaccharides (FOS) (including inulin), galactooligosaccharides (GOS), trans-galactooligosaccharides, xylooligosaccharides (XOS), chitooligosaccharides (COS), soy oligosaccharides (e.g., stachyose and raffinose) gentiooligosaccharides, isomaltooligosaccharides, mannooligosaccharides, maltooligosaccharides and mannanoligosaccharides.
  • FOS fructooligosaccharides
  • XOS galactooligosaccharides
  • COS chitooligosaccharides
  • soy oligosaccharides e.g., stachyos
  • Oligosaccharides are not necessarily single components, and can be mixtures containing oligosaccharides with different degrees of oligomerization, sometimes including the parent disaccharide and the monomeric sugars.
  • Various types of oligosaccharides are found as natural components in many common foods, including fruits, vegetables, milk, and honey.
  • Specific examples of oligosaccharides are lactulose, lactosucrose, palatinose, glycosyl sucrose, guar gum, gum Arabic, tagalose, amylose, amylopectin, pectin, xylan, and cyclodextrins.
  • Prebiotics may also be purified or chemically or enzymatically synthesized.
  • a disease associated with increased H 2 S levels comprising administering to a subject in need thereof an effective amount of the engineered microorganism described herein, wherein the microorganism consumes H 2 S.
  • the disease associated with increased H 2 S levels may be a neurodegenerative disease, a myocardial injury, an ophthalmic disease, a gastrointestinal disorder, inflammatory bowel disease (IBD), an ulcer, colorectal cancer, mental retardation, Down Syndrome, intestinal pain, H 2 S poisoning, an inflammatory disease, or diabetes mellitus.
  • IBD inflammatory bowel disease
  • provided herein are methods of treating a disease associated with decreased H 2 S levels, comprising administering to a subject in need thereof an effective amount of the engineered microorganism described herein, wherein the microorganism produces H 2 S.
  • the disease associated with decreased H 2 S levels may be a cardiovascular disease, hypertension, a kidney disease, or a retinal disease.
  • methods of treating a disease associated with changes in H 2 S levels comprising administering to a subject in need thereof an effective amount of the engineered microorganism described herein.
  • NEX-16225 changes in H 2 S levels may be neurodegenerative disease (Tripathi SJ, Chakraborty S, Miller E, Pieper AA, Paul BD. Hydrogen sulfide signalling in neurodegenerative diseases. Br J Pharmacol), a myocardial injury (Elrod JW, Calvert JW, Morrison J, et al. Hydrogen sulfide attenuates myocardial ischemia-reperfusion injury by preservation of mitochondrial function), an ophthalmic disease (Li P, Liu H, Shi X, Prokosch V.
  • Hydrogen Sulfide Novel Endogenous and Exogenous Modulator of Oxidative Stress in Retinal Degeneration Diseases. Molecules.2021;26(9):2411), a gastrointestinal disorder (Singh SB, Lin HC. Hydrogen Sulfide in Physiology and Diseases of the Digestive Tract. Microorganisms.2015;3(4):866- 889), inflammatory bowel disease (IBD) (Singh SB, Lin HC. Hydrogen Sulfide in Physiology and Diseases of the Digestive Tract. Microorganisms.2015;3(4):866-889), irritable bowel syndrome (IBS) (Singh SB, Lin HC.
  • IBD inflammatory bowel disease
  • IBS irritable bowel syndrome
  • liver fibrosis Wang DY, Li HM, Guo JC, Duan SF
  • Ji XY Hydrogen Sulfide as a Novel Regulatory Factor in Liver Health and Disease. Oxid Med Cell Longev.2019;2019:3831713
  • liver ischemia ⁇ reperfusion injury Wang DD, Wang DY, Li HM, Guo JC, Duan SF, Ji XY. Hydrogen Sulfide as a Novel Regulatory Factor in Liver Health and Disease.
  • Oxid Med Cell Longev.2019;2019:3831713 hepatotoxicity
  • Wang DD, Wang DY, Li HM, Guo JC, Duan SF Ji XY. Hydrogen Sulfide as a Novel Regulatory Factor in Liver Health and Disease.
  • Oxid Med Cell Longev.2019;2019:3831713) acute liver failure (Wu DD, Wang DY, Li HM, Guo JC, Duan SF, Ji XY. Hydrogen Sulfide as a Novel Regulatory Factor in Liver Health and Disease. Oxid Med Cell Longev. 2019;2019:3831713), osteoporosis (Grassi F, Tyagi AM, Calvert JW, et al. Hydrogen Sulfide Is a Novel Regulator of Bone Formation Implicated in the Bone Loss Induced by Estrogen Deficiency.
  • Hydrogen Sulphide-Based Therapeutics for Neurological Conditions Perspectives and Challenges. Neurochem Res.2023;48(7):1981-1996)
  • ischemic stroke Sharif AH, Iqbal M, Manhoosh B, et al. Hydrogen Sulphide-Based Therapeutics for Neurological Conditions: Perspectives and Challenges. Neurochem Res.2023;48(7):1981-1996)
  • skin diseases such as burn (Xu M, Zhang L, Song S, et al. Hydrogen sulfide: Recent progress and perspectives for the treatment of dermatological diseases. J Adv Res. 2020;27:11-17), diabetic skin wound (Xu M, Zhang L, Song S, et al.
  • Hydrogen sulfide Recent progress and perspectives for the treatment of dermatological diseases. J Adv Res. 2020;27:11-17), psoriasis (Xu M, Zhang L, Song S, et al. Hydrogen sulfide: Recent progress and perspectives for the treatment of dermatological diseases. J Adv Res.2020;27:11-17), systemic sclerosis (Xu M, Zhang L, Song S, et al. Hydrogen sulfide: Recent progress and perspectives for the treatment of dermatological diseases. J Adv Res.2020;27:11-17), melanoma (Xu M, Zhang L, Song S, et al. Hydrogen sulfide: Recent progress and perspectives for the treatment of dermatological diseases.
  • the present disclosure provides a method of reducing H 2 S levels in environmental water or wastewater, comprising administering thereof an effective amount of the engineered microorganism described herein.
  • the method is used for the environmental degradation of H 2 S. For example, using the same engineered microbes described herein for degrading H 2 S from wastewater plants or source waters used in drilling.
  • a method of delivering a pharmaceutical composition described herein to a subject is administered in conjunction with the administration of an additional therapeutic.
  • the pharmaceutical composition comprises the engineered microorganism co-formulated with the additional therapeutic.
  • the pharmaceutical composition is co-administered with the additional therapeutic.
  • the additional therapeutic is administered to the subject before administration of the pharmaceutical composition (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes before, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours before, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days before).
  • the pharmaceutical composition e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes before, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours before, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days before.
  • the additional therapeutic is administered to the subject after administration of the pharmaceutical composition (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes after, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours after, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days after).
  • the same mode of delivery are used to deliver both the pharmaceutical composition and the additional therapeutic.
  • different modes of delivery are used to administer the pharmaceutical composition and the additional therapeutic.
  • the pharmaceutical composition is administered orally while the additional therapeutic is administered via injection (e.g., an intravenous, intramuscular and/or intratumoral injection).
  • the dosage regimen can be any of a variety of methods and amounts, and can be determined by one skilled in the art according to known clinical factors. As is known in the medical arts, dosages for any one patient can depend on many factors, including the subject's species, size, body surface area, age, sex, immunocompetence, and general health, the particular microorganism to be administered, duration and route of administration, the kind and stage of the disease, for example, tumor size, and other compounds such as drugs being administered concurrently or near-concurrently. In addition to the above factors, such levels can be affected by the infectivity of the microorganism, and the nature of the microorganism, as can be determined by one skilled in the art.
  • appropriate minimum dosage levels of microorganisms can be levels sufficient for the microorganism to survive, grow and replicate.
  • the dose of the pharmaceutical compositions described herein may be appropriately set or adjusted in accordance with the dosage form, the route of administration, 106 FH12512877.6 Attorney Docket No.: NEX-16225 the degree or stage of a target disease, and the like.
  • the general effective dose of the agents may range between 0.01 mg/kg body weight/day and 1000 mg/kg body weight/day, between 0.1 mg/kg body weight/day and 1000 mg/kg body weight/day, 0.5 mg/kg body weight/day and 500 mg/kg body weight/day, 1 mg/kg body weight/day and 100 mg/kg body weight/day, or between 5 mg/kg body weight/day and 50 mg/kg body weight/day.
  • the effective dose may be 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, or 1000 mg/kg body weight/day or more, but the dose is not limited thereto.
  • the dose administered to a subject is sufficient to prevent disease (e.g., autoimmune disease, inflammatory disease, metabolic disease, cancer), delay its onset, or slow or stop its progression.
  • disease e.g., autoimmune disease, inflammatory disease, metabolic disease, cancer
  • dosage will depend upon a variety of factors including the strength of the particular compound employed, as well as the age, species, condition, and body weight of the subject.
  • the size of the dose will also be determined by the route, timing, and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound and the desired physiological effect. Suitable doses and dosage regimens can be determined by conventional range-finding techniques known to those of ordinary skill in the art.
  • treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached.
  • An effective dosage and treatment protocol can be determined by routine and conventional means, starting e.g., with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Animal studies are commonly used to determine the maximal tolerable dose ("MTD") of bioactive agent per kilogram weight. Those skilled in the art regularly extrapolate doses for efficacy, while avoiding toxicity, in other species, including humans.
  • MTD maximal tolerable dose
  • the dosages of the active agents used in accordance with the invention vary depending on the active agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage.
  • the dose should be sufficient to result in slowing, and preferably regressing, the growth of the tumors and most preferably causing complete regression of the cancer.
  • NEX-16225 Separate administrations can include any number of two or more administrations, including two, three, four, five or six administrations.
  • the methods provided herein include methods of providing to the subject one or more administrations of an pharmaceutical composition, where the number of administrations can be determined by monitoring the subject, and, based on the results of the monitoring, determining whether or not to provide one or more additional administrations. Deciding on whether or not to provide one or more additional administrations can be based on a variety of monitoring results.
  • the time period between administrations can be any of a variety of time periods.
  • the time period between administrations can be a function of any of a variety of factors, including monitoring steps, as described in relation to the number of administrations, the time period for a subject to mount an immune response and/or the time period for a subject to clear the MP from normal tissue.
  • the time period can be a function of the time period for a subject to mount an immune response; for example, the time period can be more than the time period for a subject to mount an immune response, such as more than about one week, more than about ten days, more than about two weeks, or more than about a month; in another example, the time period can be less than the time period for a subject to mount an immune response, such as less than about one week, less than about ten days, less than about two weeks, or less than about a month.
  • the time period can be a function of the time period for a subject to clear the MP from normal tissue; for example, the time period can be more than the time period for a subject to clear the MP from normal tissue, such as more than about a day, more than about two days, more than about three days, more than about five days, or more than about a week.
  • the delivery of an additional therapeutic in combination with the pharmaceutical composition described herein reduces the adverse effects and/or improves the efficacy of the additional therapeutic.
  • the effective dose of an additional therapeutic described herein is the amount of the therapeutic agent that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, with the least toxicity to the patient.
  • the effective dosage level can be identified using the methods described herein and will depend upon a variety of pharmacokinetic factors including the activity of the particular 108 FH12512877.6 Attorney Docket No.: NEX-16225 compositions administered, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • an effective dose of an additional therapy will be the amount of the therapeutic agent which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • the toxicity of an additional therapy is the level of adverse effects experienced by the subject during and following treatment.
  • Adverse events associated with additional therapy toxicity include, but are not limited to, abdominal pain, acid indigestion, acid reflux, allergic reactions, alopecia, anaphylasix, anemia, anxiety, lack of appetite, arthralgias, asthenia, ataxia, azotemia, loss of balance, bone pain, bleeding, blood clots, low blood pressure, elevated blood pressure, difficulty breathing, bronchitis, bruising, low white blood cell count, low red blood cell count, low platelet count, cardiotoxicity, cystitis, hemorrhagic cystitis, arrhythmias, heart valve disease, cardiomyopathy, coronary artery disease, cataracts, central neurotoxicity, cognitive impairment, confusion, conjunctivitis, constipation, coughing, cramping, cystitis, deep vein thrombosis, dehydration, depression, diarrhea, dizziness, dry mouth, dry skin, dyspepsia, dysp
  • the microbe senses intestinal sulfide below the engineered set-point, it turns on the sulfide production pathway. Conversely, if intestinal sulfide levels are too high, the consumption pathway will be activated.
  • the nreB/C system has not yet been used for sensing H 2 S. Further, the CstR and SqrR systems rely on sulfide oxidation followed by sensing of hydrogen polysulfides, whereas the nreB/C sensor directly senses H 2 S.
  • H 2 S Hydrogen sulfide
  • E. coli to titrate H 2 S controllably across the physiological range in a gut microphysiological system (chip) supportive of the co-culture of microbes and host cells.
  • the chip was designed to maintain H 2 S gas tension and enable visualization of co-culture in real-time with confocal microscopy.
  • pCas and pTargetF Additional plasmids were gifts from Sheng Yang.
  • a custom N20 sequence was added to pTargetF via PCR to target each gene of interest directly.
  • pCas and pTargetF were transformed and cured according to the protocol. Colonies were picked, and knockouts were verified with PCR and Sanger sequencing (Genewiz from Azenta Life Sciences). The strains were grown overnight in M9 minimal media (4 g/L glucose, 1 g/L casamino acids (cat.502 no 2240, VWR), and 1 mg/L thiamine (cat.
  • E. coli genes (decR, yhaM, yhaO) were cloned from E. coli MG1655 gDNA (cat no. T3010, New England Biolabs Inc.) via PCR.
  • the cdl gene (from F. nucleatum) was codon optimized for E. coli and synthesized (Integrated DNA Technologies). Gibson assembly (cat no. E2621, New England Biolabs Inc.) was used to assemble backbones and genes of interest. 111 FH12512877.6 Attorney Docket No.: NEX-16225 Once assembled, plasmids were chemically transformed into DH5-alpha cells (cat no. C2987, New England Biolabs Inc.) and plated on agar plates containing appropriate antibiotics. Colonies were picked and were sequence verified with PCR and Sanger sequencing. Once verified, plasmids were mini-prepped (cat no.
  • GMPS fabrication The GMPS device used in this study is a derivative of previous work from our lab. The fabrication used poly(methyl methacrylate) (PMMA) sheets, PET membranes, double- sided adhesive tape, and a laser cutter. Each layer of the chip was designed with computer- aided drafting (CAD, Autodesk Inventor) and was laser cut (Epilog Zing 16, Epilog Laser).
  • CAD computer- aided drafting
  • the 3/16” PMMA (PMMA, McMaster-Carr) contained six laser cut holes, two for inlet flow, two for outlet flow, and two for access to apical and basal channels (referred to as ‘seed ports’), which were tapped to create threading for Luer lock fittings. This piece served as the top layer of the device.
  • the second layer was a sheet of 1/16” PMMA (PMMA, McMaster- Carr) inserted between two pieces of 50 ⁇ m thick double-sided adhesive tape (966 Adhesive Transfer Tape, 3M), which served as the ceiling of the apical channel. All six circular inlets and outlets were laser-cut to match the through-holes in the top layer.
  • the third layer was a PET membrane with 0.4 ⁇ m diameter pores (ipCELLCULTURE Track Etched Membrane, it4ip S.A.) that sat between the second and fourth layers, creating a cell culture surface for the apical channel.
  • Circular inlets and outlets were laser-cut to match the through-holes in the fourth layer.
  • a sheet of 1/16” PMMA inserted between two pieces of 50 ⁇ m thick double- sided adhesive tape served as the fourth layer and the ceiling of the basal channel. Three circular inlets and outlets were laser cut to match the through-holes in the top two layers.
  • the fifth layer was a no.1 glass coverslip that sealed the basal channel. Using a custom device, the layers were pressed together while maintaining the alignment of the holes and channels.
  • the PVC tubing was connected to a syringe pump (NE-1600 Six Channel Programmable Syringe Pump, New Era Pump Systems Inc.), perfusing culture medium through the chip. Details of the GMPS can be found in a schematic in Figure 5.
  • Caco-2 cell culture in GMPS Caco-2 cells were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle Medium (DMEM, cat no. MT10013CV, Corning) supplemented with 10% fetal bovine serum (FBS, cat no.10437028, ThermoFisher) and 100 U/mL penicillin ⁇ streptomycin (cat no.15140122, ThermoFisher).
  • FBS fetal bovine serum
  • penicillin ⁇ streptomycin cat no.15140122, ThermoFisher
  • Microfluidic chips were removed from the vacuum chamber and UV sterilized (300 mJ/cm) for 10 minutes (Spectrolinker XL- 1000, Spectronics Corporation) on each side.
  • Chips were then treated with oxygen plasma (Expanded Plasma Cleaner PDC-001, Harrick Plasma) for 1 minute. Fittings and tubing were sterilized by autoclaving. The chips, fittings, tubing, and syringe pump were assembled in a biosafety cabinet using an aseptic technique. The apical channel was coated in a 400 ⁇ g/mL solution of rat tail type I collagen (cat no. 354249, Corning), after which the chips were placed in a humidified incubator at 37C and 5% CO 2 for one hour. After this time, the culture medium was perfused through the chip in preparation for seeding Caco-2.
  • oxygen plasma Exposured Plasma Cleaner PDC-001, Harrick Plasma
  • Caco-2 cells were harvested from flasks and concentrated to 5,000,000 cells/mL.200 ⁇ L of Caco-2 cells were manually injected into the chip via the apical seed port.
  • the chip set-up was placed in a humidified incubator at 37C and 5% CO 2 without flow for two hours to allow cell adhesion. After two hours, the apical and basal flow was initiated at 3 ⁇ L/minute (shear stress 0.076 dyne/cm). Effluent media was collected in waste collection tubes. Chips were under flow for seven days. On the seventh day, apical media was swapped for sterile PBS++ (pH 7.4) with 25 mM Lucifer Yellow (cat no. L453, Thermo Fisher).
  • PBS++ was made of 1 L of DPBS, no calcium, no magnesium (cat no. 14190144, Thermo Fisher), 2.4 g/L HEPES (cat no. BP310-1, Fisher Scientific), 100 g/L anhydrous calcium chloride (cat no.349615000, Thermo Fisher), and 48.4 g/L pure magnesium chloride (cat no.223210010, Thermo Fisher).
  • the basal media was swapped with antibiotic-free DMEM with 10% FBS in preparation for the inoculation of engineered strains. The media was allowed to perfuse through the chip overnight to clear residual antibiotics.
  • Caco-2 cells were between passage numbers 25 and 47.
  • Sodium sulfide nonahydrate (Na2S) (cat no.208043, Sigma Aldrich) was used for exogenous sulfide.
  • DSS (cat no. AC433240050 Fisher Scientific) was dissolved in PBS++ to 3% w/v.
  • the GMPS set-up was moved to the biosafety cabinet, and flow was paused.200 ⁇ L of bacteria were injected into the apical seed port at an OD600 of 0.4 using a syringe pump (100 ⁇ L/min). Once inoculated, the chip set-up was moved back into the humidified incubator at 37C 5% CO2, and flow was resumed immediately at 3 ⁇ L/min.
  • CFU analysis Apical and basal effluent was collected at hours -4, 18, and 42. Apical samples were serially diluted 1,000-fold or 10,000-fold, and 50 ⁇ L were plated on LB agar plates containing appropriate antibiotics or antibiotic-free plates for control chip samples. For basal samples, 50 ⁇ L of basal effluent was directly plated. Agar plates were incubated overnight at 37C and counted the following day. For apical samples, the CFU count was multiplied by a factor of 20,000 (for 1,000-fold diluted samples) or 200,000 (for 10,000-fold diluted samples) to get units of CFU/mL.
  • mBBr monobromobimane
  • the flow rate through the column was 0.5 mL/min, and a gradient elution was applied to separate thiols: 0-2 min, 0% B; 2-8 min, 46% B; 8-9 min, 64% B; 9-13 min, 100% B; 13-14 min, 0% B; 14-15 min, 0% B.
  • Solvent A was 10% methanol and 0.25% glacial acetic acid, adjusted to pH 3.9.
  • Solvent B was 90% methanol and 0.25% glacial acetic acid, adjusted to pH 3.9.
  • the protocol was adapted and modified from previously described protocols. RT-qPCR analysis After two days of co-culture, the flow was stopped, and the GMPS set-up was removed from the incubator.
  • RNA lysis buffer (cat no. T2010, New England Biolabs Inc.) was applied to the channel and vigorously pipetted. RNA was purified following the manufacturer’s protocol (cat no. T2010, New England Biolabs Inc.). Oligo(dT)12-18 (cat no.18418012, Thermo Fisher) was used to reverse transcribe Caco-2 RNA (C1000 Touch Thermal Cycler, BioRad). The resulting cDNA was cleaned and concentrated (cat no. D40144, Zymo Research) and 1 117 FH12512877.6 Attorney Docket No.: NEX-16225 ⁇ L was used for qPCR.
  • a SYBR Green-based qPCR master mix (cat no. M3003, New England Biolabs Inc.) and primers were used to amplify genes of interest (C1000 Thermal Cycler with CFX96 Real-Time System, BioRad). Primers were made with NCBI Primer Blast. The target gene, GADD45a, was normalized to GAPDH. Relative expression differences were calculated using the delta-delta Ct method. Immunofluorescent imaging in GMPS Images of tight junction formation were assessed using fluorescence microscopy.
  • Monolayers were fixed by flowing 4% formaldehyde (v/v in 2.5% Goat Serum) at 9 ⁇ L/min for 30 minutes, 0.1% TritonX (v/v in 2.5% Goat Serum) at 9 ⁇ L/min for 20 minutes, and 2.5% goat serum for 30 minutes at room temperature. Chips were covered in foil and placed in the fridge (4C) overnight. The basal channel was flushed with PBS. Flow rates were kept below 10 ⁇ L/min in the apical channel to prevent high shear stress and monolayer disruption. The following day, 1:200 ZO-1 conjugated antibody (cat no.
  • MA3-39100-A647, Thermo Fisher) and 1:10,000 Hoechst were mixed in 2.5% goat serum and flowed at 6 ⁇ L/min for one hour. After one hour, the flow was paused, and the system sat static for one hour. Afterward, the monolayers were washed with DPBS for three hours at 4 ⁇ L/min.
  • the GMPS was imaged with a confocal microscope (Zeiss LSM 880 NLO) using the DAPI and Alexa Fluor 647 filters.
  • T7-yhaM-RFP + BAD-yhaO was inoculated on the chip.1:2,000 Hoechst was added to apical and basal channels and flowed on the chip at 3 ⁇ L/min for three hours. Adding a higher concentration of Hoechst to apical and basal channels enhanced the signal, which may be attributed to the sizeable microbial population in the apical channel disrupting the mass transport of Hoechst to the Caco-2 nuclei. Before imaging, the basal channel was flushed with PBS. Next, the GMPS was imaged with confocal microscopy using the DAPI and mRFP-1.2 filters.
  • a live video of the co-culture was captured using an inverted fluorescence microscope (Zeiss Axio Observer Z1). To do this, the GMPS was kept in an incubated microscope set up (37C and 5% CO 2 ), and normal experimental conditions were maintained including perfusion through the chip. Images were captured overnight and assembled in a movie using Zen software (Zeiss) and Adobe Premier Pro. Image processing was done in Zen software (Zeiss). Gaussian smoothing was used for all images.
  • Statistical analysis A one-way ANOVA or two-way ANOVA followed by post hoc Tukey-Kramer analysis were used to determine statistical significance (*p ⁇ 0.05).
  • Bar graphs represent the 118 FH12512877.6 Attorney Docket No.: NEX-16225 average value from independent experiments, and dots represent the result from each independent experiment. Error bars represent the ⁇ standard deviation.
  • GraphPad Prism was used to carry out the analysis. Graphs were formatted in Adobe Illustrator. For each independent experiment, bacterial cultures were grown from cryostock, and Caco-2 cells were harvested from flasks. Independent experiments were performed on different days. For GMPS experiments, each independent experiment represents an individual GMPS. For samples that were lower than the limit of quantification, they were labeled as not detected (Source Data File). Gut microphysiological system (GMPS) characterization The GMPS used herein is a derivative of the work by Hosic.
  • the chip is laser cut and assembled layer-by-layer resulting in a chip with polymethyl methacrylate (PMMA) apical and basal channels separated by a polyester (PET) membrane (Fig.5A).
  • PMMA polymethyl methacrylate
  • PET polyester
  • Fig.5A Constant perfusion of culture medium was similar to physiological flow (flow rate 3 ⁇ L/minute, shear stress 0.076 dyne/cm) to mimic shear forces in the intestine.
  • a homogeneous immortalized Caco-2 epithelial monolayer populated the entire length of the PET membrane (Fig.5B).
  • Caco-2 tight junctions were validated by immunofluorescent staining for tight junction protein Zonula Occludens-1 (ZO-1) and the live nuclei stain, Hoechst (Fig.5C).
  • This GMPS is fabricated from gas impermeable PMMA which retains gas tension better than PDMS, enabling the study of H 2 S on host epithelial cells (Fig.5E). Effluent media can be used for on-chip metabolic analysis and quantifying monolayer permeability. The GMPS provides easy access to the cell culture channels enabling RNA extraction from host cells (Fig 5E). Strain development and cysteine desulfidase activity Wild-type (WT) E. coli MG1655 possesses multiple cysteine desulfidase genes and produces 70 ⁇ 41 ⁇ M and 1719 ⁇ 97 ⁇ M in non-growth (no glucose or amino acids) and growth media, respectively (Figs.6A and 6B).
  • H 2 S levels in the intestine range from 300 to 3,400 ⁇ M.
  • sseA also known as mstA
  • malY the transcription factor decR
  • the decR gene codes for the transcription factor, DecR, which is activated by cysteine and regulates the yhaOM operon in E. coli.
  • yhaO codes for the putative cysteine transporter and yhaM a cysteine desulfidase (Fig.5D).
  • the literature is divided regarding the cysteine desulfidase activity of these gene products attributable perhaps to different E. coli strains, oxygen concentrations, growth media, or sulfide detection methods used in the various studies. Since it was critical the base strain produced below 300 ⁇ M H 2 S, the decR strain was selected as the chassis strain for subsequent engineering. To develop strains capable of controllably producing higher levels of H 2 S, we next assembled a small library of plasmids with various cysteine desulfidase homologs under the control of different promoters (Fig.5D), then transformed them into the chassis strain. H 2 S production was tested in Hungate tubes – a closed system – to minimize H 2 S evaporation.
  • T7-decR in non-growth media did not show increased H 2 S production.
  • DecR is activated by cysteine, which was added when cells were in the non-growth media, limiting transcription of yhaO and yhaM.
  • Titratable microbial H 2 S production in the GMPS Next, the engineered strains were inoculated in the GMPS to test their performance and production of H 2 S in a simulated gut environment (Fig.7A).
  • the GMPS has different physical conditions than Hungate tubes, including constant perfusion and shear stress which may affect microbial gene expression and activity.
  • the Lac-cdl strain (herein named ‘low-sulfide strain’) was used to demonstrate H 2 S titration by simply changing concentrations of the inducer, IPTG.
  • the inducer IPTG.
  • NEX-16225 experimental control over inlet streams perfusing the GMPS, 5 mM cysteine, varying amounts of inducer, and antibiotic(s) for plasmid maintenance were continuously fed for the duration of the experiment. Samples were taken before strain inoculation to ensure there was no baseline H 2 S or microbial contamination in the GMPS (hour -2 in Fig.7).
  • H 2 S was measured at hours 1, 20, and 44 (Fig.7A).
  • GMPSs containing no bacteria and only 5 mM cysteine, 250 ⁇ M IPTG, and chloramphenicol served as the negative control, confirming Caco-2 cells are unable to convert cysteine into significant levels of H 2 S.
  • the low-sulfide strain produced across a 3.5-fold range (74 – 262 ⁇ M H2S) by varying inducer concentrations from 0, 2.5, and 250 ⁇ M IPTG (Fig.7B). These strains permit a high degree of control over the lower range of H 2 S levels in the chip, enabling investigation of how small changes in H 2 S levels impact host biology.
  • T7-yhaM + BAD-yhaO (herein named ‘high-sulfide strain’) was inoculated on the chip.
  • the high-sulfide strain maximized H 2 S production on the chip, producing 1,216 ⁇ M H 2 S (Fig. 7C).
  • the lower production of H 2 S on chip compared to Hungate tubes could be explained by some evaporation during sample collection, or alteration of transcription or activity resulting from the constant perfusion and associated shear stress activity.
  • E. coli cells in the effluent over two days it was plausible E. coli was colonizing the chip.
  • the apical channel contained non- growth media and cysteine, and in Hungate tubes, cysteine alone was insufficient to support growth.
  • Previous work in GoC systems demonstrated the enterorecirculation phenomenon, a physiological process in which basal metabolites diffuse into the apical lumen, making it possible glucose could diffuse through the Caco-2 monolayer into the apical channel for E. coli utilization (Fig.8A). To test this, we measured the levels of glucose and E.
  • coli metabolites (acetate, formate, succinate) in apical effluent collected before and after inoculation of E. coli, and from a negative control chip with no microbe inoculation.
  • glucose was present in the apical channel at ⁇ 0.1 g/L.
  • negative control chips no acetate was found, and glucose levels stayed constant for the duration of the experiment.
  • glucose fell to undetectable levels, and acetate was produced at ⁇ 0.1 g/L.
  • the GMPS was under flow which implies there was constant basal-to-apical glucose flux, and glucose was undetectable after microbial inoculation, indicating that E.
  • coli actively metabolized glucose for two days in the co-culture (Fig.8B).
  • a chip inoculated with the high-sulfide strain engineered to constitutively express red fluorescent protein (RFP) was imaged by confocal microscopy (Fig.8C and 8D).
  • a video taken with an inverted microscope shows the GMPS was colonized by WT E. coli MG1655 expressing RFP under flow conditions for multiple hours (Fig.8E). Imaging confirms these strains maintain a population on a chip under constant flow, without washout, for the duration of the experiment (two days).
  • coli is known to be able convert thiosulfate through rhodaneses to sulfite and sulfide.
  • thiosulfate production in a chip perfused with Na 2 S and inoculated with E. coli, but without addition of cysteine (i.e., no microbial H 2 S production).
  • H 2 S is a known genotoxic agent and has been shown to change the transcriptional activity of various Caco-2 genes like growth arrest and DNA damage- inducible alpha (GADD45a).
  • GADD45a growth arrest and DNA damage- inducible alpha
  • those experiments were conducted in static in vitro cultures with exogenous H 2 S sources.
  • Caco-2 cells co- cultured with the high-sulfide strain for two days had a 6.2-fold increase in GADD45a expression compared to control (no sulfide).
  • This effect is likely mediated by H 2 S-induced DNA damage leading to cell cycle arrest, and supports the hypothesis that H 2 S affects the host in a concentration-dependent manner.
  • the effect of exogenous Na 2 S on GADD45a expression is more variable than the high and low-sulfide strains, with coefficients of variation of 0.45, 0.32, and 0.14, respectively, highlighting the increased precision in dosing achievable by an engineered microbial source of H 2 S.
  • Example 3 H 2 S production from different sources and in complex in vitro systems and animals
  • Five human fecal samples were acquired. Samples were taken from cryo preservation and moved to an anoxic chamber and the cultures were inoculated in brain heart infusion media in air-tight serum bottles to maintain anaerobic conditions. Cultures were moved to a 124 FH12512877.6 Attorney Docket No.: NEX-16225 37C shake incubator (200 RPM) for overnight growth. After growth, the cultures were moved to an anoxic chamber to be handled under anaerobic conditions.
  • the culture was centrifuged at 16,000 RCF and resuspended in an anaerobic minimal glucose media, M9.1 mM of each sulfur substrate was added and the cultures were placed in Hungate tubes to maintain anaerobic conditions and preserve H 2 S gas tension.
  • the cultures were moved to a 37C shake incubator (200 RPM) and H 2 S was measured using the methylene blue assay at 0, 3, and 20 hours after incubation.
  • CTX103 was taken from cryo preservation and inoculated in LB media and grown until stationary phase.300 uL of saturated culture was inoculated in 15 mL LB media supplemented with 100 uM Isopropyl ⁇ - d-1-thiogalactopyranoside (IPTG) and 10 mM L- arabinose to induce gene expression in a 125 mL Erlenmeyer flask. The cells were cultured overnight in a 20C shake incubator (200 RPM). After growth, the cultures were moved to an anoxic chamber to be handled under anaerobic conditions.
  • IPTG Isopropyl ⁇ - d-1-thiogalactopyranoside
  • the culture was centrifuged at 16,000 RCF and resuspended in an anaerobic minimal glucose media, M9.1 mM of glutathione was added, and the cultures were placed in Hungate tubes to maintain anaerobic conditions and preserve H 2 S gas tension.
  • the cultures were moved to a 37C shake incubator (200 RPM) and H 2 S was measured using the methylene blue assay at 0, 3, and 20 hours after incubation.
  • Fig.15 we wanted to understand how different sulfur compounds are metabolized to H 2 S by the human gut microbiota. To begin, we acquired 5 human fecal microbiome samples.
  • CTX103 produces H 2 S from GSH under monoculture and in co-culture with human gut microbiota derived from human stool, under anaerobic conditions (Fig.17).
  • 125 FH12512877.6 Attorney Docket No.: NEX-16225 A truncated version of CTX103 which was published in Cell Reports (Hayes JA, et al.
  • CTX104 was taken from cryo preservation and inoculated in LB media and grown until stationary phase in a 37C shake incubator (200 RPM).300 uL of saturated culture was inoculated in 15 mL LB media in a 125 mL Erlenmeyer flask. After two hours, 10 mM L- arabinose was added to express relevant genes.
  • the culture was centrifuged at 16,000 RCF and moved to an anoxic chamber and resuspended in anaerobic M9 media.1 mM of sodium sulfite was added, and the cultures were placed in Hungate tubes to maintain anaerobic conditions and preserve H 2 S gas tension. The cultures were moved to a 37C shake incubator (200 RPM) and H 2 S was measured using the methylene blue assay at 0, 3, and 20 hours after incubation. We have also gathered triplicate data for our strain, CTX104, which converts sulfite into H 2 S.
  • the engineered cell expresses the sulfite reductase subunits, cysI and cysJ, to reduce sulfite to H 2 S (Fig.26).
  • cysI contains one siroheme and to compensate, CTX104 also expresses the siroheme synthetase,cysG.
  • CTX104 was tested in anaerobic M9 media supplemented with 1 mM sodium sulfite (Fig.27).
  • Example 4 H 2 S consumption It is well established that H 2 S plays a role in several gut pathologies. Developing a microbe to degrade H 2 S would have tremendous value for patients. Based on the data in Fig.
  • CTX101 which only differs by expressing the homologous gshA-gshB ligases to synthesize GSH. These genes are contained on the pACYCDuet-1 plasmid and driven by the T7 promoter.
  • CTX101, CTX102, and empty vector cells were taken from cryo preservation and inoculated in LB media and grown until stationary phase in a 37C shake incubator (200 RPM). 300 uL of saturated culture was inoculated in 15 mL LB media supplemented with 100 uM IPTG to induce gene expression in a 125 mL Erlenmeyer flask. The cells were cultured overnight in a 20C shake incubator (200 RPM). After growth, the cultures were moved to an anoxic chamber to be handled under anaerobic conditions.
  • the culture was centrifuged at 16,000 RCF and resuspended in an anaerobic minimal glucose media, M9.1.5 mM of H 2 S, 2 mM L-serine, 20 mM glycine, and 20 mM glutamic acid was added and the cultures were placed in Hungate tubes to maintain anaerobic conditions and preserve H 2 S gas tension.
  • the cultures were moved to a 37C shake incubator (200 RPM) and H 2 S was measured using the methylene blue assay at 0, 3, and 20 hours after incubation.100 uL of each culture was taken, centrifuged at 16,000 RCF, and supernatant was collected and stored at -80C until quantification with monobromobimane and HPLC.
  • CTX101/CTX102 consume H 2 S and produce cysteine and GSH under anaerobic conditions (Fig.20). This is critical because the gut is mostly anoxic. Notably, we engineered different pathways which result in different H 2 S consumption and cysteine/GSH production levels (CTX101 vs CTX102). GSH synthesis in the engineered cells is significantly higher than in the wild type (WT). It is notable that Fig.15 shows GSH should not be converted to H 2 S by the native human microbiome. CTX101/102 will degrade toxic H 2 S to produce GSH, which will be stable and not converted back to H 2 S by the native gut microbiota.
  • CTX102 or an empty vector control of CTX102, were orally gavaged to Sprague- Dawley rats (see Fig.21 description for experimental details). Although the study was not powered to identify statistically significant changes, small intestinal (SI) and colonic H 2 S levels decreased by 72% and 38%, respectively, and warrants further investigation (Fig.21). Notably, inflammatory bowel diseases patients have fecal H 2 S levels 30% higher than healthy patients (Gibson GR, Cummings JH, Macfarlane GT), signifying CTX102 may reduce clinically relevant levels of H 2 S. Based on our discovery that GSH is not converted to H 2 S by most gut microbiota, we developed a novel therapeutic.
  • CTX111 and CTX112 were taken from cryo preservation and inoculated in LB media and grown until stationary phase in a 37C shake incubator (200 RPM).300 uL of saturated culture was inoculated in 15 mL LB media in a 125 mL Erlenmeyer flask. After two hours, 10 mM L-arabinose was added to express relevant genes.
  • the cultures were centrifuged at 16,000 RCF and moved to an anoxic chamber and resuspended in anaerobic LB media.1.2 mM of H 2 S, 20 mM nitrate, and 20 mM fumarate was added, and the cultures were placed in Hungate tubes to maintain anaerobic conditions and preserve H 2 S gas tension. The cultures were moved to a 37C shake incubator (200 RPM) and H 2 S was measured using the methylene blue assay at 0, 3, and 20 hours after incubation.100 uL of each culture was taken, centrifuged at 16,000 RCF, and supernatant was collected and stored at -80C until quantification with monobromobimane and HPLC.
  • CTX111 and CTX112 in Escherichia coli S1030, which express the sulfide quinone reductase (SQR) enzymes from Rhodobacter Capsulatus and Wolinella succinogenes, respectively as shown in Fig.23.
  • SQR sulfide quinone reductase
  • H 2 S hydrogen sulfide
  • Fig.24 shows that CTX111 and CTX112 degrade H 2 S under anaerobic conditions. The figure caption describes the details of the experiment.
  • CTX101/102 and CTX111/112 which degrade H 2 S by two different mechanisms of action.
  • Attorney Docket No.: NEX-16225 in vivo It is novel that we can combine these strains as an engineered bacterial consortium to degrade H 2 S by two unique mechanisms of action.
  • CTX112 was taken from cryo preservation and inoculated in LB media and grown until stationary phase in a 37C shake incubator (200 RPM).300 uL of saturated culture was inoculated in 15 mL LB media in a 125 mL Erlenmeyer flask.
  • CTX112 was cultured with HD-6 in different cell density ratios as measured by OD 600 , 1:1, 10:1, and 100:1 CTX112:HD-6, respectively.
  • CTX112 consumes all H 2 S after 3 hours at 10:1 and 100:1 cell ratio, and after 20 hours at 1:1 cell ratio (Fig.25).
  • the enzyme sulfide quinone reductase (SQR), converts H 2 S to polysulfides and they serve as a stable sulfur sink in human fecal consortia.
  • SQR sulfide quinone reductase
  • Example 5 Sense-and-respond strains An H 2 S sensor from B. lichiniformis to sense H 2 S and mount a transcriptional response to consume H 2 S may be used.
  • a sense-and respond system which is using a known thiosulfate sensor as our sensor (Daeffler KN, Galley JD, Sheth RU, et al., Mol Syst Biol. 2017;13(4):923. Published 2017 Apr 3).
  • the sensor contains a 2-component sensing system which sense extracellular thiosulfate and activates a transcription factor which binds a promoter and drives gene expression of green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • strain 129 FH12512877.6 Attorney Docket No.: NEX-16225 will sense thiosulfate levels and mount a transcriptional response for H 2 S consumption that is proportional to the thiosulfate level.
  • the sense-and-respond strain was grown from cryopreservation and grown in LB media overnight in a 37C shake incubator (200 RPM).300 uL of saturated culture was inoculated in 15 mL LB media in a 125 mL Erlenmeyer flask and grown for two hours. After two hours, 100 uM IPTG and varying concentrations of thiosulfate (0, 0.01, 0.1, 1 mM thiosulfate) were added to cultures and induced for two hours.
  • the cultures were centrifuged at 16,000 RCF and resuspended in aerobic LB media containing 1.5 mM H 2 S, and the cultures were placed in Hungate tubes to maintain H 2 S gas tension.
  • the cultures were moved to a 37C shake incubator (200 RPM) and H 2 S was measured using the methylene blue assay at 0, 3, and 20 hours after incubation.100 uL of each culture was taken, centrifuged at 16,000 RCF, and supernatant was collected and stored at -80C until quantification with monobromobimane and HPLC.
  • Fig.22 shows that the strain senses the disease biomarker, thiosulfate, and responds by turning on a proportional transcriptional response in H 2 S consumption and cysteine production in a concentration-dependent manner. It is novel to develop an engineered bacteria that senses a disease biomarker, thiosulfate, and turns on a proportional transcriptional response for a therapeutic purpose. The transcriptional response is expression of the H 2 S degradation pathway highlighted in Fig.19. This innovation is novel and makes bacteria-based therapeutics more targeted and precise.
  • our sense-and-respond bacteria will mount a therapeutic response (i.e., amount of H 2 S degraded) based on the severity of the disease state (i.e., amount of thiosulfate present in the environment).
  • a therapeutic response i.e., amount of H 2 S degraded
  • Example 6 Methods Methylene Blue Assay for H 2 S Quantification: 200 uL of the experimental sample was added to 615 mL zinc acetate mixture (600 mL of 1% w/v zinc acetate dihydrate with 15 mL 3 M sodium hydroxide) and vortexed. After 130 FH12512877.6 Attorney Docket No.: NEX-16225 5–10 min of incubating at room temperature, 150 mL of 0.1% n-n-dimethylethylenediamine in 5 M hydrochloric acid was added, followed by 150 mL of 23 mM ferric chloride in 1 M hydrochloric acid.

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Abstract

L'invention concerne des micro-organismes, des systèmes et des procédés de modulation de H2S. Dans certains aspects, la présente divulgation concerne des micro-organismes génétiquement modifiés capables de consommer du H2S, de convertir le H2S en L-cystéine, la L-cystéine en glutathion, ou en persulfures, polysulfures ou agrégats de soufre. Dans certains aspects, la présente divulgation concerne des micro-organismes génétiquement modifiés capables de produire du H2S, de dégrader le glutathion en L-cystéine et H2S, ou de réduire le sulfite en H2S. Dans certains aspects, la présente divulgation concerne des micro-organismes génétiquement modifiés capables de détecter des signaux environnementaux associés à un état pathologique et d'activer des voies de consommation de H2S.
PCT/US2024/049315 2023-09-29 2024-09-30 Procédés pour modulation de niveaux in vivo de sulfure d'hydrogène par des organismes modifiés Pending WO2025072960A2 (fr)

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