Nickel-chelating compounds, compositions, and methods for inhibiting methanogenesis and other nickel-dependent biological processes

Abstract

The present disclosure relates to methods and compositions for inhibiting methanogenesis by reducing bioavailable nickel in a biological environment. Methanogenic microorganisms require nickel for the activity of key enzymes, including methyl coenzyme M reductase, and reduction of nickel availability below biologically required levels disrupts methanogenic metabolism and methane production. In certain embodiments, methanogenesis is inhibited by administering a nickel chelator in an amount effective to reduce bioavailable nickel to sub-micromolar or nanomolar concentrations. Nickel chelators may include protease-resistant nickel-binding peptides capable of functioning in protease-rich or anaerobic environments. Such peptides may comprise structural features conferring protease resistance, including incorporation of D-amino acids, non-canonical amino acids, α,α-disubstituted amino acids, cyclization, or cyclotide scaffolds. The disclosed methods and compositions are applicable to ruminant animals and other anaerobic systems and may be implemented using synthetic formulations or biological expression in microorganisms or plants.

Description

[0001] PATENT Docket No. 0719.000001 WOOl

[0002] N1CKEL-CHELAT1NG COMPOUNDS, COMPOSITIONS, AND METHODS FOR INHIBITING METHANOGENESIS AND OTHER NICKEL-DEPENDENT BIOLOGICAL PROCESSES

[0003]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004]

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application 63 / 746,367, filed January 17, 2025, and U.S. Provisional Patent Application 63 / 808,951, filed May 20, 2025, each of which is incorporated by reference herein in its entirety.

[0005]

[0003] INCORPORATION BY REFERENCE

[0006]

[0004] All publications, patents, and patent applications referenced in this specification are incorporated by reference to the same extent as if each were specifically and individually incorporated by reference. To the extent any incorporated reference contradicts this disclosure, this specification controls.

[0007]

[0005] TECHNICAL FIELD

[0008]

[0006] The present disclosure relates to metal-chelating compositions, including nickel-selective chelators and nickel-binding peptides, and their use in inhibiting methanogenesis (including enteric methanogenesis in ruminants and biogenic methane generation in other environments) and in modulating other nickel-dependent biological processes, including processes mediated by nickel metalloenzymes such as methyl coenzyme M reductase (MCR) and urease. In certain embodiments, “nickel-dependent biological processes” further include processes mediated by one or more of: NiFe-hydrogenases, carbon monoxide dehydrogenase / acetyl-CoA synthase, nickel superoxide dismutase, lactate racemase, acireductone dioxygenase, and / or nickeldependent uptake / transport / trafficking pathways (including NikABCDE-type and NiCoT-type transport and metallochaperone-mediated delivery).

[0009]

[0007] BACKGROUND

[0008] Methane is a potent greenhouse gas that contributes to climate change. A significant portion of global methane emissions arises from biogenic methane, including enteric methane produced by ruminant animals (e.g., cattle, sheep, goats) as well as methane produced in anoxic or anaerobic environments such as wetlands, rice paddies, manure treatment systems, landfills, and biodigesters. There remains a significant and unmet need for methods and compositions that reduce methane production across these settings.

[0010]

[0009] In ruminant production systems, methanogenesis represents not only an environmental issue but also a loss of feed efficiency as methane formation diverts carbon and hydrogen away from productive animal outputs. Effective mitigation strategies ideally reduce methane production while maintaining or improving feed efficiency and / or animal performance.

[0011]

[0010] Methanogenesis is catalyzed by archaeal methanogens and is believed to depend universally on methyl coenzyme M reductase (MCR). MCR requires the nickel-containing cofactor F430, and methanogens exhibit a uniquely high biological demand for nickel. In view of methanogens’ dependence on nickel for F430 biosynthesis and MCR function, the inventors recognized that reducing the bioavailability of nickel in target environments could provide a broad and mechanistically targeted method to inhibit methanogenesis.

[0012]

[0011] While certain inhibitors of methanogenesis act via direct inhibition or inactivation of MCR, practical limitations may include the need for continuous administration, metabolic instability, potential toxicity concerns, and / or microbial adaptation. In contrast, nickel deprivation targets an upstream and essential nutrient requirement of methanogens and can be implemented through multiple modalities (e g., small-molecule chelators, engineered peptides, sorbents / polymers, biological chelators, and feed or environmental strategies).

[0013]

[0012] However, chelators suitable for real-world biological environments must overcome challenges including insufficient nickel specificity, insufficient binding affinity, and poor stability in protease-rich or otherwise reactive environments (e.g., rumen fluid). Accordingly, there is a need for nickel-chelating compositions that are sufficiently selective and stable for use in demanding biological matrices.

[0014]

[0013] SUMMARY

[0015]

[0014] Described herein are methods and compositions for inhibiting methanogenesis and for modulating other nickel-dependent biological processes by reducing the bioavailability of nickel in a target environment.

[0015] In one aspect, the disclosure provides a method for inhibiting methanogenesis in a target environment (including, without limitation, the rumen of a ruminant or in rice paddies), comprising administering to the target environment an effective amount of a nickel chelator to reduce the bioavailability of nickel, thereby inhibiting methanogen growth and / or methane production.

[0016]

[0016] In another aspect, the disclosure provides compositions comprising nickel chelators suitable for administration to ruminants as a feed additive, top-dress, total mixed ration (TMR) component, engineered expression in forage crops, water-dosed composition, mineral supplement, bolus, slow-release capsule, nutraceutical, or pharmaceutical composition.

[0017]

[0017] In another aspect, the disclosure provides protease-resistant nickel-binding peptides that exhibit high affinity and / or specificity for nickel and retain activity in protease-rich environments. In certain embodiments, protease resistance is achieved by one or more of (i) incorporation of D-amino acids (including full enantiomeric peptides), (ii) incorporation of non-canonical amino acids, including P-amino acids, (iii) incorporation of a,a-disubstituted residues, and / or (iv) cyclization (including head-to-tail, sidechain-to-sidechain, or head-to-sidechain cyclization).

[0018]

[0018] In another aspect, the disclosure provides methods for screening nickel-binding peptides for desired properties including: nickel-dependent inhibition of methanogen growth and / or methane production; specificity demonstrated via nickel “rescue”; the demonstration of antifouling properties that prevent aggregation and adsorption in complex biological environments by incubation in media containing sterile rumen fluid, and stability demonstrated by retention of functional activity following protease exposure or incubation in rumen fluid.

[0019]

[0019] In another aspect, the disclosure provides additional nickel-chelation modalities that may be used alone or in combination with peptide embodiments, including: small-molecule chelators that precipitate upon nickel binding; nickel(II) ion-imprinted polymers; nickel-binding proteins or complexes; DNA aptamers; and low-nickel feeds and / or hyperaccumulating plant-based strategies.

[0020]

[0020] In another aspect, the disclosure provides combination methods comprising administering a nickel chelator together with (a) a methane inhibitor (e.g., an MCR inhibitor), (b) an acetogenic microorganism, (c) one or more non-nickel elements or minerals, and / or (d) a masking agent to increase palatability.

[0021] In another aspect, the disclosure provides methods comprising: (i) measuring total nickel and / or bioavailable nickel in the target environment (e.g., rumen fluid, manure slurry, sediment porewater), (ii) administering a nickel chelator in an amount selected based on the measured nickel level, and (iii) optionally repeating measurement and adjusting administration to maintain bioavailable nickel below a target threshold associated with reduced methanogenesis.

[0021]

[0022] In another aspect, the disclosure provides kits comprising (a) a nickel chelator composition described herein and (b) instructions for use to reduce methanogenesis and / or modulate a nickel-dependent biological process, optionally together with (c) a nickel measurement reagent or sampling device, (d) a rumen-retained delivery device, and / or (e) a mineral supplementation composition.

[0022]

[0023] Examples are described below and will be incorporated following the Detailed Description, including data for both a small molecule (DMG) and a number of nickel-binding peptides that demonstrate nickel chelation, methanogen growth inhibition, reduction in methane production and nickel-dependent inhibition of methanogen growth. Further experimental results will teach methods to develop protease resistant peptides that achieve significantly enhanced stability in rumen fluid relative to naturally occurring peptides.

[0023]

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0024]

[0025] FIG. 1 is a photograph of tubes showing red Ni-DMG complex formation in a 70% clarified rumen fluid matrix, where addition of 0.5 mM DMG (tubes #3 and #4) or 1 mM DMG (tubes #5 and #6) resulted in a red complex that is readily visualized compared to tubes without DMG (tubes #1, #2, #7, and #8).

[0025]

[0026] FIG. 2 is a graph showing that dimethylglyoxime (DMG) inhibits methanogen growth (M. maripaludis) in a dose-dependent manner. Average and SE (A=3 to 5) of optical density at 600 nm (ODeoo) are presented in the figure. M. maripaludis cultures were grown in McFc basal medium, with added nickel (N) and dimethylglyoxime (DMG) concentrations in pM, as indicated in the inset. These data show that at a dose of 250 pM DMG does not significantly inhibit AT. maripaludis growth, but that doses of at least 500 pM show marked inhibition of growth.

[0027] FIG. 3 is a graph showing that DMG inhibits methanogen growth in a nickel -dependent manner, Average and SE (N=3 to 5) of optical density at 600 nm (ODeoo) are presented in the figure. M. maripaludis cultures were grown in McFc basal medium, with added nickel (N) and DMG concentrations in pM, as indicated in the right inset. N250DMG250 achieves growth similar to N0DMG0, showing that with excess nickel, DMG does not inhibit growth. reporting ODeoo (average and SE; N=3 to 5) for M. maripaludis grown in McFc basal medium with nickel and DMG concentrations (pM) as indicated, where a condition with excess nickel (N250DMG250) achieves growth similar to N0DMG0, indicating rescue with nickel.

[0026]

[0028] FIG. 4 is a bar plot showing that DMG reduces methane production in the chemical environment of the rumen. Average and standard deviation of triplicate measurements of methane accumulated in culture headspace are presented, expressed as percentage normalized to methane accumulation in plain McFc media (100%). Cells were grown in basal McFc (left panel) or McFc-70% rumen fluid hybrid medium (right panel). Conditions (DMG or nickel supplementation) are indicated underneath each column. These results show a clear dose and nickel dependent methane production in both McFc and McFc modified to include 70% rumen fluid.

[0027]

[0029] FIG. 5 A-F are tables showing the results from representative peptides which were screened for growth inhibition activity against M. maripaludis often at one or more concentrations and timepoints. % inhibition means the reduction of growth as measured by the optical density at 600 nm relative to a control condition in which no peptide is added.

[0028]

[0030] FIG. 6 A-D are tables showing the results from representative peptides which were evaluated in secondary screening to determine: (1) dose-dependent inhibition; (2) nickel-rescue behavior; and (3) maintenance or loss of inhibition in the presence of competitive metals (e.g., Zn, Cu). In this Figure, Peptide Cone means the concentration of peptide tested. Ni Cone means the concentration of added nickel that was tested. Zn Cone means the concentration of added Zinc that was tested. TP1 means the first time point and is measured in hours. TP2 means the second time point and is measured in hours. % Inhib means % inhibition of growth as measured by the optical density at 600 nm relative to a control condition in which no peptide or metal are added.

[0029]

[0031] FIG 7. is a graph showing that SEQ ID NO: 140 reduces methanogen growth in a dose and nickel-dependent manner in McFc media, with less zinc-dependence. Average and SE (A=2to 3) of optical density at 600 nm (ODeoo) vs. metal condition, faceted by peptide concentration and hours post inoculation (hpi). M. maripaludis cultures were grown in McFc basal medium, with added nickel (Ni), zinc (Zn) and nickel chelating peptide SEQ ID NO: 140. These data show that SEQ ID NO: 140 inhibits M. maripaludis growth in a dose dependent manner, and that growth can be rescued by addition of excess nickel, but markedly less by addition of excess zinc.

[0030]

[0032] FIG. 8 A-F is a table showing representative results from the PAR binding assay described in example 4. Candidate peptides were tested in the PAR-Ni competition assay across a peptide concentration series. Results are reported as A500 values and / or derived % Inhibition, referring to the A500 value measured from a control condition in which peptide is not added to compete with PAR for binding. Peptide cone, means the concentration of peptide added to the condition.

[0031]

[0033] DETAILED DESCRIPTION

[0032] 1. Definitions and Interpretation

[0033]

[0034] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0034]

[0035] As used herein, “nickel chelator” refers to any molecule, polymer, peptide, protein, complex, aptamer, sorbent, or other chemical or biological material or composition that binds nickel (e.g., Ni(II)). A nickel chelator may reduce the bioavailable nickel in a target environment. A nickel chelator may bind nickel reversibly or irreversibly and may form soluble complexes or insoluble complexes (including precipitates).

[0035]

[0036] As used herein, “nickel-selective” (or “nickel-specific”) refers to preferential binding of nickel relative to at least one competing metal present in a target environment (e.g., Zn(II), Cu(II), Co(II), Fe( 11 / 111 ), Mn(II)), as demonstrated by one or more of: (i) a measurable reduction in free and / or bioavailable nickel in the presence of competing metals, (ii) retention of functional inhibition of methanogenesis in the presence of competing metals, and / or (iii) competition assays indicating at least about 10x, 50x, 100*, or 1000* preference for nickel under assay conditions relevant to the target environment.

[0036]

[0037] As used herein, “nickel rescue” refers to reversal or partial reversal of a nickel-chelator-induced phenotype (e.g., growth inhibition and / or methane reduction) upon addition ofexogenous nickel in an amount sufficient to exceed the nickel-binding capacity of the chelator under the tested conditions.

[0037]

[0038] As used herein, “bioavailable nickel” refers to forms of nickel in a target environment that are accessible for biological uptake, transport, or utilization by organisms (including methanogens) for nickel-dependent processes (including F430 biosynthesis and MCR function) or nickel metalloenzymes (including urease). Reducing bioavailable nickel may include binding dissolved nickel, sequestering nickel, precipitating nickel, or otherwise lowering nickel activity.

[0038]

[0039] In certain embodiments, “dissolved nickel” refers to nickel present in a filtrate obtained by filtering a sample through a pore size of about 0.45 pm or about 0.2 pm.

[0039]

[0040] In certain embodiments, total and / or dissolved nickel is quantified by ICP-MS, atomic absorption spectroscopy, electropotentiometric methods, or colorimetric methods. In certain embodiments, “bioavailable nickel” is estimated using chelator competition assays, diffusive gradients in thin films (DGT), and / or speciation modeling to estimate free Ni(II) activity.

[0040]

[0041] As used herein, “methanogenesis” refers to biological methane production, including but not limited to archaeal methanogenesis in rumen and non-rumen environments.

[0041]

[0042] As used herein, “target environment” refers to any biological or engineered environment in which methanogenesis and / or other nickel-dependent biological processes occur. Non-limiting examples include: a rumen, reticulum, or other forestomach compartment of a ruminant; manure systems; anaerobic digesters; wetlands; rice paddies; landfills; and other anoxic environments.

[0042]

[0043] As used herein, “protease-resistant” refers to a peptide or peptide-containing composition exhibiting increased stability against proteolytic degradation relative to a corresponding all-L linear, canonical peptide under relevant conditions (e.g., in rumen fluid and / or in the presence of one or more proteases).

[0043]

[0044] As used herein, “effective amount” refers to an amount sufficient to achieve a desired outcome, such as reducing methanogen growth, reducing methane production, reducing methane emissions, shifting fermentation away from methanogenesis, increasing volatile fatty acid production, increasing feed efficiency, improving animal performance, or increasing tolerance to nonprotein nitrogen sources.

[0044]

[0045] As used herein, qualitative descriptors of methanogen growth inhibition based on optical density measurements (e.g., ODeoo) correspond to the following quantitative ranges. “No inhibition” refers to a condition in which the average OD«» of replicate cultures (e.g., N = 3)incubated in the presence of a test peptide is from about 90% to about 100% of the average ODsoo of corresponding control cultures incubated in the absence of the peptide, corresponding to from about 0% to about 10% inhibition relative to control. “Partial inhibition” refers to a condition in which the average ODsoo of replicate cultures (e.g., N = 3) incubated in the presence of a test peptide is from about 50% to less than about 90% of the average ODsoo of corresponding control cultures, corresponding to greater than about 10% to less than about 50% inhibition relative to control. “Strong inhibition” refers to a condition in which the average ODsoo of replicate cultures (eg., N = 3) incubated in the presence of a test peptide is from about 0% to about 50% of the average ODsoo of corresponding control cultures, corresponding to from about 50% to about 100% inhibition relative to control.

[0045]

[0046] In certain embodiments, “a level insufficient to support methanogenic activity” refers to a dissolved and / or bioavailable nickel concentration below a threshold associated with impaired methanogen growth and / or methane production, including below about 8 pM, 2 pM, 1 pM, 500 nM, 100 nM, 50 nM, or 20 nM, and may be confirmed by nickel rescue as described herein.

[0046]

[0047] As used herein, amino acids and / or amino acid residues may be referred to using their full name, their conventional three letter abbreviation, or their conventional one letter abbreviation. When using the one letter abbreviation, capital letters indicate L-amino acids or residues, and lower-case letters indicate D-amino acids or residues. For example, arginine may be referred to as Arg, R, or r. Various amino acids and their abbreviations are listed in Table 1.

[0047]

[0048]

[0049]

[0048] Examples of P-amino acids include but are not limited to P-alanine and P-aminobutyric acid. Examples of N-methylated amino acids include but are not limited to N-m ethylthreonine, N-methylalanine, and sarcosine (N-methylglycine).

[0050]

[0049] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0051]

[0050] As used in this specification and the appended claims, the term "or" is generally employed in its sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means one or all of the listed elements or a combination of any two or more of the listedelements. The use of "and / or" in some instances does not imply that the use of "or" in other instances may not mean "and / or."

[0052]

[0051] The words "preferred" and "preferably" refer to embodiments of the disclosure that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure.

[0053]

[0052] As used herein, "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising" or the like are used in their open ended inclusive sense, and generally mean "include, but not limited to," "includes, but not limited to," or "including, but not limited to."

[0054]

[0053] It is understood that wherever embodiments are described herein with the language "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising" and the like, otherwise analogous embodiments described in terms of "consisting of' and / or "consisting essentially of are also provided. The term "consisting of means including, and limited to, whatever follows the phrase "consisting of." That is, "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. The term "consisting essentially of indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0055]

[0054] Reference throughout this specification to "one embodiment," "an embodiment," "certain embodiments," or "some embodiments," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0056]

[0055] Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure.Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0057]

[0056] In the description herein particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.

[0058]

[0057] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0059]

[0058] 2. Rationale: Nickel Deprivation as a Control Point for Methanogenesis

[0060]

[0059] Methanogenesis depends on MCR and its nickel-containing cofactor F430. Methanogens may require higher nickel concentrations for growth than other nickel-utilizing organisms, creating an exploitable vulnerability: nickel limited growth and metabolic activity.

[0061]

[0060] The inventors have recognized that bioavailable nickel functions as a shared upstream control variable across diverse anaerobic target environments, including animal gastrointestinal systems, agricultural waste handling systems, and engineered anaerobic digesters. Accordingly, the disclosed methods are applicable to any target environment in which nickel-dependent biological processes occur and are not limited to administration within a living animal.

[0062]

[0061] In certain embodiments, administration of a nickel chelator reduces dissolved nickel in a rumen from a naturally occurring baseline (e.g., -micromolar levels) to levels insufficient for high-nickel-requiring methanogens. In certain embodiments, dissolved nickel is reduced below about 8 pM, below about 2 pM, below about 1 pM, below about 500 nM, below about 100 nM, below about 50 nM, or below about 20 nM, thereby inhibiting methanogens while allowing growth of competing microorganisms such as acetogens that may tolerate lower nickel availability. In one embodiment, dissolved nickel is not detectable.

[0062] In certain embodiments, effective inhibition of methanogenesis is achieved by reducing dissolved or bioavailable nickel to sub-micromolar concentrations, including nanomolar concentration ranges. Methanogenic archaea exhibit an unusually high dependence on nickel due to the requirement for nickel-containing cofactors, including coenzyme F430, which is essential for methyl coenzyme M reductase activity. As reported in the literature, biosynthesis and functional activity of such nickel-dependent cofactors are highly sensitive to nickel availability, and limitation of nickel to nanomolar levels has been shown to impair methanogenic metabolism even in the presence of otherwise sufficient nutrients.

[0063]

[0063] Accordingly, in some embodiments, nickel chelators described herein reduce bioavailable nickel to concentrations below about 1 micromolar, and in certain embodiments to concentrations below about 100 nanomolar, thereby limiting availability of nickel for methanogenic microorganisms. Reduction of bioavailable nickel to such levels may occur locally within a biological environment, including microenvironments within the rumen or other anaerobic systems, and does not require complete removal of total nickel from the system.

[0064] Rather, functional nickel deprivation sufficient to disrupt methanogenic activity may be achieved by sequestration of nickel into forms that are not accessible for uptake or utilization by methanogens.

[0065]

[0064] Nickel depletion to sub-micromolar or nanomolar levels may be inferred from functional assays described herein, including inhibition of methane production and reversal of inhibition upon supplementation with excess nickel, as well as from competition-based nickel-binding assays. Such reductions in bioavailable nickel are consistent with known nickel requirements of methanogens and provide a mechanistic basis for the observed inhibition of methanogenesis.

[0066]

[0065] As used herein, reduction of “bioavailable nickel” does not necessarily require direct chelation of nickel in a classical coordination chemistry sense. Nickel may be rendered biologically inaccessible by any mechanism that reduces its availability for uptake, trafficking, or utilization by methanogenic microorganisms, including sequestration, precipitation, immobilization, compartmentalization, misdirection into non-productive pools, interference with transport or metallochaperone-mediated delivery, or formation of nickel complexes that are not substrates for biological nickel acquisition systems. Accordingly, references herein to “nickel chelators” and “nickel chelation” encompass compositions and methods that functionally deprivemethanogens of biologically accessible nickel, regardless of the specific physicochemical mechanism by which such deprivation is achieved.

[0067]

[0066] In some embodiments, nickel deprivation shifts rumen fermentation away from methane formation toward alternative hydrogen sinks such as acetogenesis and / or propionate production, thereby supporting or improving feed efficiency and animal performance. Such shifts may be evidenced by increased flux through acetogenic pathways, increased propionate formation, reduced hydrogen partial pressure, or altered relative abundance or activity of hydrogen-utilizing microbial populations.

[0068]

[0067] In certain embodiments, nickel chelation is configured to be pH-sensitive such that nickel binding is strong at ruminal pH (approximately pH 5-7) but substantially reduced at lower pH values encountered downstream in the gastrointestinal tract (e.g., abomasal pH of approximately pH 2-4). In such embodiments, nickel and / or other metals bound to the chelator in the rumen are released upon transit to lower-pH compartments, thereby localizing nickel deprivation to the rumen while reducing systemic metal depletion. In some embodiments supplemental metal or minerals can be provided alongside the nickel chelator in order to ensure that any off-target binding to non-nickel metals or minerals does not deplete the bioavailability of these materials.

[0069]

[0068] In certain embodiments, pH-sensitive binding is achieved by selecting nickel-binding chelators with metal-coordinating ligands whose donor atoms are protonated at low pH (thereby weakening metal coordination), including amine- and imidazole-containing ligands, and / or by incorporating acid-labile linkages or coatings that expose or release nickel-binding functionality primarily within a ruminal pH window. In certain embodiments, nickel binding is characterized at two or more pH values (e.g., pH ~6 and pH ~2-4) and the chelator is selected to exhibit at least about a 10*, 50*, or 100* reduction in nickel-binding affinity and / or capacity at the lower pH relative to ruminal pH.

[0070]

[0069] In these embodiments, nickel deprivation is functionally selective and spatially localized, such that nickel availability is reduced to levels insufficient to support methanogenic activity within the rumen while avoiding systemic depletion of nickel or other essential trace elements required for host physiology.

[0071]

[0070] 3. Compositions: Nickel Chelator

[0072]

[0071] Nickel chelators suitable for use herein include, without limitation:

[0072] In certain embodiments, nickel chelators are selected to satisfy one or more performance criteria relevant to the target environment, including: (i) sufficient nickel-binding affinity to reduce bioavailable nickel below a threshold associated with reduced methanogenesis, (ii) nickel selectivity in the presence of competing metals at concentrations present in the target environment, (iii) chemical stability under relevant pH and redox conditions, (iv) functional stability in the presence of proteases (for peptide embodiments), and (v) compatibility with formulation and delivery constraints (e.g., feed processing, storage, rumen retention, and / or controlled release). In certain embodiments, a nickel chelator is provided as an insoluble or immobilized form (e.g., supported chelators, resins, functionalized clays, or rumen-retained devices) to localize nickel sequestration within the target environment and reduce systemic absorption.

[0073]

[0073] In certain embodiments, the small-molecule chelator comprises a vicinal dioxime nickel-binding scaffold (dioxime), including substituted glyoximes and / or cyclic dioximes, and / or functional analogs thereof selected to form a high-stability Ni(II) complex (optionally a low-solubility complex) under target-environment conditions. In certain embodiments, the smallmolecule chelator is administered as a pro-chelator that is converted in situ (e.g., by pH change, enzymatic conversion, or redox conditions) into an active nickel-binding form. In certain embodiments, small-molecule chelators are immobilized on an insoluble carrier (e.g., polymer beads, silica, cellulose, clays, biochar) to reduce systemic absorption and to localize nickel sequestration within the target environment. In certain embodiments, the small-molecule chelator is selected to exhibit pH-dependent nickel binding such that nickel complexation is favored at ruminal pH and disfavored at lower pH downstream, thereby releasing bound metals after rumen transit.

[0074]

[0074] In certain embodiments, the ion-imprinted polymer is prepared by polymerizing one or more functional monomers capable of coordinating Ni(II) (e.g., vinyl imidazole, methacrylic acid, vinyl pyridine, acrylamide, thiol-functional monomers) in the presence of Ni(II) as a template, together with a crosslinker (e.g., ethylene glycol dimethacrylate or other di- / multi-functional crosslinkers) and a porogen to form a porous polymer. After polymerization, Ni(II) is removed (e.g., by washing with acid, chelator, or salt solutions) to generate nickel -selective binding sites. In certain embodiments, the ion-imprinted polymer is provided as particles,granules, monoliths, coatings, or porous matrices configured to be rumen-retained and / or to provide controlled nickel-binding capacity over time.

[0075]

[0075] In certain embodiments, nickel-binding proteins or complexes are immobilized on an insoluble support (e.g., silica, cellulose, polymer beads) or provided as non-viable biomass (e.g., heat-killed or fixed microbial cells expressing a nickel-binding protein) to function as a nickel sorbent in a target environment while limiting absorption or systemic distribution.

[0076]

[0076] In certain embodiments, the disclosure provides compositions comprising a nickel-binding material, wherein the nickel-binding material is configured to reduce bioavailable nickel in a rumen of a ruminant animal to a level insufficient to support methanogenic activity. In some embodiments, the reduction of bioavailable nickel occurs through sequestration, precipitation, immobilization, or functional inaccessibility of nickel, independent of whether the nickel-binding material forms a soluble nickel complex.

[0077]

[0077] In certain embodiments, the nickel-binding material comprises a small-molecule chelator, an ion-imprinted polymer, a functionalized solid support, a rumen-retained device, and / or a precipitating nickel complex. In some embodiments, the nickel-binding material is insoluble or becomes insoluble upon binding nickel.

[0078]

[0078] In certain embodiments, nickel-binding aptamers are identified or optimized using SELEX-based selection against Ni(II) presented in a desired chemical form (e.g., Ni(II) salts, Ni(II) complexes, Ni(II)-functionalized resins), and optionally counter-selected against competing metals to improve selectivity. In certain embodiments, aptamers comprise stabilizing modifications to improve persistence in biological matrices, including phosphorothioate backbones, 2'-O-methyl, 2'-fluoro, locked nucleic acids (LNA), and / or conjugation to carriers or particles to reduce nuclease degradation and / or localize activity.

[0079]

[0079] In certain embodiments, nickel chelators described herein (including aptamers, peptides, polymers, or other nickel-binding materials) are identified or optimized from diverse libraries generated using a variety of approaches, including chemical synthesis, biological expression, mutagenesis, combinatorial assembly, or computational design. Library diversity may be introduced through, without limitation, randomization, directed variation, error-prone replication, transcription or translation, recombination, or in silico sequence or structure generation.

[0080]

[0080] In certain embodiments, selection or screening of candidate nickel chelators is performed using functional, biochemical, or physicochemical assays, including binding assays, competitionassays, precipitation or immobilization assays, chromatographic separation, differential retention or elution, or biological activity assays such as nickel-dependent inhibition of methanogenesis or nickel-rescue experiments. Selection or screening may be iterative and may be conducted in vitro, in silico, in vivo, or in hybrid workflows combining computational prediction with experimental validation.

[0081]

[0081] 3.1 Small-Molecule Nickel Chelators

[0082]

[0082] In some embodiments, the nickel chelator comprises a small molecule selected to have affinity and / or selectivity for nickel. In certain embodiments, the small-molecule chelator is dimethylglyoxime (DMG) (including salts, hydrates, solvates, and isotopic forms). DMG is soluble in an unbound form and forms a nickel complex that can be less soluble and / or precipitate, thereby sequestering nickel and reducing its bioavailability. In certain embodiments, the nickel chelator is selected from DMG and other sulfur- and / or nitrogen-donor ligands, including mercapto-acrylic acids, dithiocarbamates, thiol-containing chelators, and related ligands. Experimental data in the Examples demonstrate nickel binding and functional inhibition of methanogen growth and methane output using DMG, including nickel-dependent rescue experiments and dose-dependent effects on methanogen growth and methane production.

[0083]

[0083] 3.2 Nickel(II) Ion-Imprinted Polymers (IIPs)

[0084]

[0084] In some embodiments, the nickel chelator comprises a nickel(II) ion-imprinted polymer configured to preferentially bind nickel ions from a target environment. In certain embodiments, the polymer is formed using nickel(II) as a template during polymerization and is configured to absorb soluble nickel and reduce bioavailability for methanogens. In certain embodiments, IIPs may be incorporated into a durable bolus or delivery device for sustained sequestration of nickel in a rumen environment.

[0085]

[0085] 3.3 Nickel -Binding Proteins and Protein Complexes

[0086]

[0086] In some embodiments, the nickel chelator comprises a nickel-binding protein or complex, including but not limited to metalloregulators and chaperones (e.g., HypA, HypB, UreE, SlyD, CooT, NikR, Nur, and related proteins or engineered homologs), and complexes comprising multiple nickel-binding subunits, and nickel-binding cofactors such as F430. Such proteins or complexes may function by sequestering nickel, competing for nickel acquisition, interfering with nickel trafficking or delivery, or otherwise reducing the biological accessibility of nickel required for methanogenic activity.

[0087] The following examples of nickel-binding proteins, complexes, and motifs are provided for illustrative purposes only and are not intended to define or limit the structural, chemical, or mechanistic features by which nickel may be bound, sequestered, or rendered biologically inaccessible in accordance with the present disclosure.

[0087]

[0088] In certain embodiments, the nickel-binding peptides described herein are short peptides or oligopeptides having a total length selected to balance metal-binding affinity, protease resistance, and bioavailability in protease-rich environments such as rumen fluid. In various embodiments, suitable peptides comprise from about 3 to about 20 amino acid residues. Shorter peptides (e.g., 3-8 or 3-12 residues) may provide compact nickel-binding motifs with reduced conformational flexibility and reduced susceptibility to proteolytic cleavage, while longer peptides (e.g., up to about 16 or up to about 20 residues) may incorporate additional residues that stabilize metal coordination geometry, enhance resistance to enzymatic degradation, or improve functional persistence in biological environments. Peptides shorter than about 3 residues generally lack sufficient functional groups for stable nickel coordination, while peptides substantially longer than about 20 residues may exhibit increased protease susceptibility or reduced selectivity. Accordingly, peptide lengths including 3-5, 3-8, 3-12, 3-16, and 3-20 amino acid residues represent non-limiting embodiments of the invention.

[0088]

[0089] In general, nickel-binding motifs suitable for use in the present disclosure comprise appropriately positioned donor atoms capable of coordinating Ni(II), including sulfur, nitrogen, and / or oxygen donors, arranged in a structural context that permits formation of a stable nickel complex. Such motifs may be defined by conserved chemical functionality rather than exact amino acid sequence and may adopt a variety of coordination environments depending on donor composition, spacing, and three-dimensional arrangement. Using these general principles, nickel-binding peptides or proteins may be designed to mimic natural nickel-binding sites or to create entirely new binding motifs not found in nature, provided that the resulting motif functionally reduces biologically available nickel.

[0089]

[0090] In certain embodiments, nickel-binding motifs are inspired by metallochaperones such as HypB, which utilize cysteine-rich sequences to coordinate nickel via thiolate donor atoms.

[0090] Exemplary motifs include di- or tri-cysteine arrangements (e.g., Cys-Xn-Cys or Cys-Xn-Cys- Yn-Cys), optionally involving a backbone or terminal amine as an additional ligand. Variationsin residue identity, spacing, stereochemistry, or structural constraint may be introduced to modulate affinity, selectivity, or stability while preserving functional nickel-binding activity.

[0091]

[0091] In certain embodiments, a “HypB motif’ refers to a cysteine-containing amino acid sequence comprising Cys-Xn-Cys, where n is an integer of 1 or greater, and X represents any amino acid residue unless otherwise specified. In some embodiments, the two cysteine residues define a dithiol coordination site capable of binding divalent metal ions such as nickel (II). In other embodiments, n is 1-10, 1-6, 1-4 or 1-2. In some embodiments, X comprises Threonine, allo-threonine, Serine, Valine or a- Aminobutyric acid residues. In some embodiments, threonine positions in such motifs are substituted with allo-threonine while preserving inhibitory activity, whereas N-methylated threonine analogs at corresponding positions may reduce inhibitory activity in certain sequence contexts. In other embodiments, the sequence comprises Cys-Xn-Cys-Yn-Cys, where n is an integer of 1 or greater, and X and Y represent any amino acid residue unless otherwise specified. In other embodiments, X comprises Threonine, allo-threonine, Serine, Valine or a-Aminobutyric acid residues and Y comprises Glycine, Alanine, Sarcosine or P-Alanine. In some embodiments, the three cysteine residues define a trithiol coordination site capable of binding divalent metal ions such as nickel(II). In other embodiments, n is 1-10, 1-6, 1-4 or 1-2.

[0092]

[0092] In certain embodiments, nickel-binding motifs are inspired by amino-terminal metalbinding motifs (e.g., ATCUN-like motifs), in which anN-terminal amine, backbone amide nitrogens, and a side-chain nitrogen donor cooperate to bind nickel. Such motifs may be implemented at the N-terminus of a peptide or protein or incorporated into engineered scaffolds that reproduce the functional nickel -binding characteristics of these sites.

[0093]

[0093] In further embodiments, nickel-binding motifs are synthetic or engineered motifs not found in naturally occurring proteins, including cyclic peptides, constrained loops, backbone-assisted motifs, or mixed donor sets comprising combinations of nitrogen, oxygen, and sulfur donors. Such motifs may be identified or optimized through computational modeling, machine learning-based design, empirical screening, combinatorial library selection, or rational design, and may be tailored for desired properties including affinity, selectivity, stability in biological matrices, manufacturability, or cost.

[0094]

[0094] 3.4 Nucleic Acid Chelators (Aptamers)

[0095] In some embodiments, the nickel chelator comprises a DNA or RNA aptamer capable of binding nickel, including sequences identified as nickel-binding aptamers.

[0095]

[0096] 3.5 Plant-Derived and Feed-Based Nickel Reduction

[0096]

[0097] In some embodiments, nickel bioavailability is reduced via a low-nickel feed and / or by cultivating or preparing feed components using low-nickel soil or via hyperaccumulator-based strategies. In certain embodiments, a field may be preconditioned by growing a nickel hyperaccumulator plant (e.g., Odontarrhena chalcidica) harvesting and removing the hyperaccumulator biomass to reduce soil nickel, and subsequently growing feed components with reduced nickel content. In certain embodiments, “substantially nickel free” refers to feed compositions having nickel below about 10 ppb, and “nickel free” refers to nickel below about 10 ppt.

[0097]

[0098] In certain embodiments, “substantially nickel free” and / or “nickel free” refer to nickel concentrations below a selected threshold and / or below a method-specific limit of detection and / or limit of quantitation for an analytical method used to measure nickel (e.g., ICP-MS), optionally after acid digestion of the sample.

[0098]

[0099] 3.6 Nickel -binding peptides

[0099]

[0100] In some embodiments, the nickel chelator comprises a short peptide capable of binding nickel, including peptides which achieve enhanced protease-resistance via use of non-canonical amino acids, D-amino acids, incorporation of a,a-disubstituted residues, cyclization and / or use of a cyclotide scaffold.

[0100]

[0101] For example, in some embodiments, peptides retain methanogen-inhibiting activity despite incorporation of non-canonical residues within the HypB-derived motif itself, including allo-threonine substitutions that preserve inhibition (e.g., SEQ ID NO:285 and SEQ ID NO:289) and a,a-disubstituted residues such as Aib and / or P-amino acids such as -Ala as extensions (e.g., SEQ ID NO:281-283 and SEQ ID NO:286-287), whereas N-methylation at a threonine position can reduce or eliminate inhibition (e.g., SEQ ID NO:284 and SEQ ID NO:288), providing guidance on substitutions that may be less preferred.

[0101]

[0102] In certain embodiments, the peptide has a length of about 3-60 amino acids (e.g., 5-30 amino acids) and comprises nickel-coordinating residues (e.g., Cys, His, Asp / Glu and / or non-canonical donor residues). In certain embodiments, the peptide is configured to present a free N-terminal amine that participates in nickel coordination, and N-terminal modifications are selectedto preserve nickel-binding function. In certain embodiments, the term “non-canonical” as used herein encompasses “non-canonical” amino acids.

[0102]

[0103] 3.7 Expression of Nickel-Binding Peptides in Biological Delivery or Production Systems

[0104] In certain embodiments, nickel -binding peptides described herein are produced in situ in a target environment through biological expression systems, thereby reducing or eliminating the need for chemical peptide synthesis and lowering manufacturing and delivery costs. Such embodiments are particularly advantageous for large-scale agricultural applications.

[0103]

[0105] In some embodiments, nickel -binding peptides are expressed by engineered probiotic microorganisms suitable for administration to animals, including ruminants. Non-limiting examples of suitable host organisms include bacteria commonly used as probiotics or feed additives, such as species of Lactobacillus, Bacillus, Enterococcus, Bifidobacterium, Propionibacterium, cyanobacteria species such as Arthrospira or yeast species such as Saccharomyces. The engineered microorganisms may be designed to express one or more nickel-binding peptides intracellularly or to secrete the peptides into the surrounding environment, including the rumen. In certain embodiments, expression occurs constitutively or is induced under conditions present in the rumen, such as anaerobiosis, pH, or nutrient availability.

[0104] Expression of protease-resistant nickel-binding peptides by probiotic microorganisms enables sustained local reduction of bioavailable nickel while minimizing systemic exposure and dosing frequency. In certain embodiments, engineered microorganisms are configured for transient passage through the gastrointestinal tract without permanent colonization, while in other embodiments microorganisms are adapted to persist or colonize for extended periods, thereby providing sustained local production of nickel-binding peptides.

[0105]

[0106] In certain embodiments, engineered microorganisms comprise biocontainment features, including auxotrophy, regulated kill-switch circuits, and / or inducible expression systems configured to limit persistence outside the host and / or outside controlled agricultural settings.

[0106]

[0107] In other embodiments, nickel -binding peptides are expressed in plants, including forage crops and cereal crops consumed by ruminants. Non-limiting examples include com, sorghum, alfalfa, ryegrass, barley, wheat, oats, triticale, and silage crops. In such embodiments, plants are engineered to express nickel-binding peptides in edible tissues, including leaves, stems, or seeds, such that ingestion of the plant material delivers the nickel-binding peptides to the rumen.

[0107] Expression may be targeted to specific plant tissues or cellular compartments to enhance peptidestability during harvest, storage, and digestion. In certain embodiments, expression is configured to occur at levels sufficient to reduce bioavailable nickel in the rumen upon normal consumption of the plant material, without adversely affecting plant growth or nutritional value. In certain embodiments, expressed nickel-binding peptides are selected or engineered to retain functional activity following post-harvest processing, including drying, ensiling, pelleting, or storage, such that ingestion of processed plant material delivers active nickel-chelating peptides to the rumen.

[0108]

[0108] In both microbial and plant-based expression embodiments, the expressed peptides may comprise protease-resistant designs as described herein, including incorporation of D-amino acids, non-canonical amino acids, a,a-disubstituted residues, cyclized structures or cyclotides. In some embodiments, the peptides are further modified or fused to carrier sequences, signal peptides, or stabilizing domains to enhance expression, secretion, persistence, or functional activity in the target environment. In some embodiments, microbial or plant-based expression systems are used to manufacture nickel-binding peptides, which are subsequently isolated, partially purified, or formulated for administration as feed additives or supplements. In certain embodiments, biological expression systems are configured to express two or more distinct nickel-binding peptides simultaneously. In certain embodiments, plants expressing nickel-binding peptides are deployed in non-agricultural environments, including wetlands or remediation sites, to reduce bioavailable nickel and associated methane emissions.

[0109]

[0109] In certain embodiments involving biological expression, protease resistance is achieved via ribosomally accessible designs (e.g., cyclization, cyclotide scaffolds, disulfide-rich scaffolds, and / or secretion / fusion strategies) and / or via post-translational modification pathways capable of introducing non-canonical features. In certain embodiments, D-amino-acid-containing peptides are produced ex vivo (e.g., by chemical synthesis) and formulated for administration, while biologically expressed embodiments utilize L-amino-acid sequences and other stability strategies, unless a host system comprises a pathway capable of introducing D-residues post-translationally and / or via non-ribosomal peptide synthesis.

[0110]

[0110] Biological expression of nickel-binding peptides enables continuous or semi-continuous delivery of nickel-chelating activity, thereby providing sustained inhibition of methanogenesis and other nickel -dependent biological processes. Such approaches are expected to significantly reduce production and formulation costs relative to purified peptide administration, whilemaintaining effective control of bioavailable nickel in complex biological systems such as the rumen.

[0111]

[0111] 3.8 Inhibition of Nickel Uptake, Transport, and Trafficking

[0112]

[0112] In certain embodiments, inhibition of methanogenesis and other nickel-dependent biological processes is achieved by interfering with biological uptake, transport, trafficking, and / or intracellular utilization of nickel, in addition to or instead of extracellular nickel chelation. In such embodiments, effective inhibition of methanogenesis arises from functional deprivation of biologically accessible nickel rather than from any requirement that nickel be bound in a particular chemical form.

[0113]

[0113] Methanogenic microorganisms rely on specialized nickel acquisition and handling systems to support biosynthesis and function of nickel -dep endent cofactors and enzymes, including coenzyme F430 required for methyl coenzyme M reductase (MCR). Such systems include membrane-associated nickel transporters, periplasmic or extracellular nickel-binding proteins, cytosolic metallochaperones, and accessory or regulatory proteins that together mediate nickel import, distribution, storage, and delivery to target enzymes.

[0114]

[0114] In certain embodiments, the compositions and methods described herein inhibit nickel uptake by methanogens by rendering nickel inaccessible to nickel transport systems. In some embodiments, this is achieved by forming nickel complexes that are not substrates for transport (e g., complexes that are sterically bulky, insoluble, membrane-impermeable, or otherwise incompatible with nickel permeases or ABC-type nickel transporters, including NikABCDE-type and NiCoT-type systems).

[0115]

[0115] In other embodiments, inhibition of nickel uptake or trafficking is achieved by compounds that interfere with nickel transport or handling pathways without directly chelating nickel. Such compounds may include, without limitation: (i) molecules that competitively inhibit nickel transporters or periplasmic nickel-binding proteins, (ii) molecules that disrupt proteinprotein interactions required for nickel handoff or delivery, (iii) molecules that misdirect nickel into non-productive intracellular pools, (iv) decoy ligands or macromolecules that sequester nickel at sites inaccessible to biosynthetic pathways, and / or (v) regulators or regulatory mimetics that downregulate or dysregulate expression or function of nickel uptake and trafficking machinery.

[0116] In certain embodiments, inhibition of nickel trafficking includes interference with metallochaperones and accessory proteins involved in nickel handling and delivery, including HypA, HypB, UreE, SlyD, CooT, and related homologs, thereby reducing delivery of nickel to nickel-dependent biosynthetic pathways, including coenzyme F430 biosynthesis.

[0116]

[0117] In certain embodiments, inhibition of nickel uptake or trafficking results in intracellular nickel levels, speciation, or localization that are insufficient to support methanogenic metabolism, even where total environmental or cellular nickel remains detectable.

[0117]

[0118] In certain embodiments, inhibition of nickel uptake or trafficking acts synergistically with extracellular nickel chelation, such that partial chelation combined with transport or trafficking interference achieves effective methanogenesis inhibition at lower chelator concentrations than would be required by chelation alone.

[0118]

[0119] In certain embodiments, inhibition of nickel uptake or trafficking is confirmed by functional assays, including nickel-rescue experiments in which addition of excess bioavailable nickel reverses inhibition, thereby demonstrating that the observed effect is mediated by limitation of biologically accessible nickel rather than non-specific toxicity.

[0119]

[0120] 4. Protease-Resistant Nickel-Binding Peptides

[0120]

[0121] 4.1 Overview

[0121]

[0122] In certain embodiments, the nickel chelator comprises a synthetic nickel-binding peptide engineered to combine:

[0122]

[0123] Nickel affinity sufficient to reduce bioavailable nickel;

[0123]

[0124] Nickel specificity relative to competing metals (e g., Zn, Cu, Co, Fe);

[0124] Stability in protease-rich environments (e.g., rumen fluid), including resistance to enzymatic degradation;

[0125] Functional activity, such as inhibiting methanogen growth and / or methane production; and / or

[0125] Anti -fouling properties to prevent aggregation and adsorption in complex biological environments.

[0126]

[0126] While peptides can be engineered for strong metal binding, many canonical L-amino-acid peptides are rapidly degraded in relevant environments such as the rumen. Surprisingly, the inventors have found that incorporation of D-amino acids, non-canonical residues, cyclization, and / or a,a-disubstituted residues can yield nickel-binding peptides that are able to bind nickelwith sufficient affinity and specificity to inhibit methanogenesis while retaining sufficient stability for practical use.

[0127]

[0127] The inventors discovered that nickel-binding affinity alone is insufficient to achieve functional inhibition of methanogenesis in protease-rich biological environments, and that effective activity imposes additional, non-obvious constraints relating to peptide stereochemistry, stability, resistance to degradation, and lack of aggregation or non-specific fouling in target environments that are not predictable from nickel-binding motifs identified under non-biological conditions.

[0128]

[0128] 4.2 Peptide Design Principles and Motifs

[0129] Nickel-binding peptides may be designed based on known nickel-coordination chemistries, including coordination geometries utilizing donor atoms from histidine, cysteine, acidic residues, and non-canonical donor residues. Non-limiting strategies include:

[0130] Incorporation of cysteine- and histidine-rich motifs;

[0131] ATCUN-like motifs and N-terminus coordination strategies;

[0132] Maintenance of an N-terminal amine (and avoidance of N-terminal capping) where the N-terminus participates in the nickel coordination sphere;

[0133] Use of thiolate ligands, including stabilized thiols;

[0134] Use of bidentate aromatic donors (e.g., bipyridyl-containing residues);

[0135] Incorporation of residues that promote irreversible or highly stable nickel capture;

[0136] Incorporation of residues that promote preferred Ni-selective geometries of coordinating ligands, including for example

[0137] i. the square planar S2N2 coordination geometry used in HypB; and / or

[0138] ii. SN3, S2N2, and SN2(O) ligand sets; and / or

[0139] Incorporation of residues that reduce fouling at target site, including for example

[0140] i. replacing residues that promote non-specific binding interactions; and / or

[0141] ii. replacing hydrophobic side-chains with neutral or hydrophilic side-chains.

[0142]

[0129] In certain embodiments, nickel binding by the peptides described herein is mediated, at least in part, by coordination through sulfur-containing amino acid residues. Cysteine residues, and other amino acids comprising thiol or thioether functional groups, are known to form strong coordination interactions with nickel ions and are commonly involved in nickel-binding motifsfound in native nickel -dependent proteins. For example, nickel-binding proteins such as HypB, UreE, and NikR utilize cysteine residues to coordinate nickel in defined geometries suitable for biological function.

[0143]

[0130] Accordingly, in some embodiments, nickel-binding peptides comprise one or more cysteine residues positioned to enable thiolate coordination of nickel. In certain embodiments, sulfur-containing residues contribute to formation of square-planar or related coordination geometries associated with high-affinity nickel binding. In addition to cysteine, structurally related sulfur-containing amino acids may be incorporated to modulate nickel-binding properties. Such residues include penicillamine, which comprises a thiol functional group and increased steric substitution relative to cysteine and may enhance metal-binding affinity, selectivity, or stability. Substitution of cysteine with penicillamine or other sulfur-containing non-canonical amino acids may be employed to preserve sul fur-mediated coordination chemistry while modifying protease susceptibility, steric environment, or peptide stability.

[0144]

[0131] In certain embodiments, nickel binding by the peptides described herein may additionally or alternatively involve coordination through nitrogen-containing amino acid residues, including histidine. The imidazole side chain of histidine is known to participate in coordination of nickel ions and is commonly observed as a ligand in native nickel -binding proteins, often in combination with sulfur-containing residues such as cysteine. In such contexts, histidine residues may contribute to stabilization of nickel -binding geometry, modulation of binding affinity, or tuning of metal selectivity.

[0145]

[0132] Accordingly, in some embodiments, nickel -binding peptides comprise one or more histidine residues positioned to enable imidazole coordination of nickel, either independently or in combination with cysteine-mediated sulfur coordination. In certain embodiments, mixed sulfur-nitrogen coordination environments may be employed to balance binding strength, reversibility, or structural constraints while preserving effective nickel sequestration.

[0146] Incorporation or substitution of histidine residues may therefore be used to adjust nickel-binding properties without relying exclusively on thiol-based coordination.

[0147]

[0133] 4.3 Protease-Resistance Strategies

[0148]

[0134] Protease resistance may be conferred by one or more of:

[0149] D-enantiomer substitution, including fully D-peptides or partial D substitutions;

[0150] Retro-enantiomer design approaches;Cyclization, including:

[0151] a. head-to-tail cyclization,

[0152] b. disulfide cyclization,

[0153] c. lactam bridges,

[0154] d. thioether cyclization, and / or

[0155] e. head-to-sidechain cyclization;

[0156] Non-canonical amino acids, including P-amino acids, N-methylated residues, penicillamine, dehydroalanine, and other non-proteinogenic residues;

[0157] a,a-disubstituted residues (e.g., Aib) to reduce protease susceptibility and increase conformational stability;

[0158]

[0135] EGylation or conjugation strategies to alter pharmacokinetics and / or persistence in the target environment.

[0159]

[0136] Peptoid (N-substituted glycine) residues and / or backbone-modified peptidomimetics that retain nickel-binding geometry while reducing protease recognition; and conformational constraint strategies such as hydrocarbon “stapling,” sidechain crosslinking, and / or backbone macrocyclization that reduce protease susceptibility.

[0160]

[0137] Utilization of cyclotide scaffolds to deliver nickel-binding motifs within a protease resistant construct

[0161]

[0138] The Examples include functional stability data in which an all-L peptide loses activity after incubation in fresh rumen fluid, while the corresponding all-D enantiomer retains inhibitory activity after extended incubation.

[0162]

[0139] 4.4 Cyclotide-Based Nickel-Binding Peptides

[0163] In certain embodiments, protease-resistant nickel-binding peptides described herein are implemented using cyclotide-based scaffolds. Cyclotides are a class of small, cyclic peptides characterized by a head-to-tail cyclized backbone and a cystine knot motif formed by multiple disulfide bonds. This structural arrangement confers exceptional stability against proteolytic degradation, thermal denaturation, and chemical degradation, including resistance to gastrointestinal and rumen-associated proteases.

[0164]

[0140] Non-limiting examples of cyclotide scaffolds include kalata-type and trypsin-inhibitor-type cyclotides. In certain embodiments, nickel-binding motifs are grafted into one or more surface-exposed loops of a cyclotide scaffold while maintaining a cyclic backbone and cystine-knot connectivity, thereby providing a stable framework that presents nickel-coordinating residues in a defined geometry.

[0165]

[0141] In some embodiments, a cyclotide scaffold is modified to incorporate one or more nickel-binding motifs, including residues capable of coordinating nickel through nitrogen-, sulfur-, or oxy gen-containing side chains. Such motifs may include histidine residues, cysteine residues, acidic residues, or combinations thereof positioned within one or more surface-exposed loops of the cyclotide framework. In certain embodiments, native cyclotide sequences are substituted or engineered to introduce nickel-binding ligands while preserving the cyclic backbone and disulfide connectivity that confer protease resistance.

[0166]

[0142] In some embodiments, nickel -binding cyclotides are designed such that nickel coordination occurs through square-planar or pseudo-square-planar geometries, including N2S2, N3S, or mixed donor sets, thereby conferring high affinity and selectivity for nickel(II). In certain embodiments, the nickel-binding cyclotide exhibits functional nickel sequestration sufficient to reduce bioavailable nickel in a biological environment containing methanogenic microorganisms.

[0167]

[0143] Cyclotide-based nickel-binding peptides may be produced synthetically or via biological expression systems. In certain embodiments, cyclotides are expressed in plants using endogenous cyclization and folding machinery, including systems that support post-translational cyclization and disulfide bond formation, eliminating the need for chemical cyclization steps. Such plant-expressed cyclotides may be delivered to ruminants through consumption of forage or cereal crops expressing the cyclotide-based nickel-binding peptide. In other embodiments, cyclotidebased peptides are expressed in microbial systems engineered to support cyclic peptide production and secretion. In certain embodiments, microbial expression systems are engineered to support production, folding, and secretion of cyclotide-based nickel-binding peptides, including through use of auxiliary folding or cyclization pathways.

[0168]

[0144] The use of cyclotide scaffolds enables incorporation of nickel-binding functionality into an exceptionally stable peptide framework, thereby enhancing resistance to proteolysis in complex biological environments such as the rumen while maintaining high-affinity nickel binding. Cyclotide-based embodiments are therefore particularly well suited for sustained nickel deprivation and inhibition of methanogenesis in agricultural settings.

[0169]

[0145] 4.5 Representative Peptide Sequences and Variants

[0146] In certain embodiments, nickel-binding peptides comprise sequences selected from the group consisting of sequences disclosed herein in FIG. 5 (in Example 2 below). By way of nonlimiting example, certain peptide families include short cysteine-containing motifs and variants incorporating D-residues, P-residues, a, a-di substituted residues, PEGmoieties, and / or cyclization elements.

[0170]

[0147] In certain embodiments, variants include conservative substitutions that preserve nickel-coordinating residues, truncations or extensions that preserve a nickel -binding core motif, and / or variants having at least about 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to a listed sequence while retaining nickel-binding function. In certain embodiments, FIG. 5 includes sequence identity numbers for representative nickel-binding peptides that (i) inhibit methanogen growth and / or methane production in a nickel-dependent manner and (ii) retain activity after protease exposure and / or rumen-fluid incubation.

[0171]

[0148] Any of the sequences described or referenced herein may include one or more amino acid residues that comprise a D-enantiomer of the corresponding amino acid.

[0172]

[0149] 4.6 Non-Fouling and Low-Aggregation Peptide Design

[0173]

[0150] In complex biological environments such as rumen fluid, peptides may be subject not only to proteolytic degradation but also to loss of functional availability due to aggregation, selfassociation, or non-specific interactions with proteins, polysaccharides, lipids, particulate matter, or microbial biomass. Such fouling or aggregation can reduce effective peptide concentration, limit access to target metals, or otherwise diminish functional activity. Accordingly, in certain embodiments, nickel-binding peptides described herein are designed to exhibit low aggregation propensity and reduced non-specific interactions under biologically relevant conditions.

[0174]

[0151] In some embodiments, low-fouling behavior is achieved through control of overall peptide architecture, including avoidance of extended hydrophobic segments, amphipathic patterns that promote self-association, or sequence motifs prone to ordered intermolecular packing. Peptides may be designed to remain predominantly soluble and dispersed in aqueous environments, even in the presence of complex biological components.

[0175]

[0152] In other embodiments, aggregation propensity is reduced by limiting formation of ordered secondary structures, including P-sheet-rich conformations associated with peptide selfassembly. Sequence designs that disrupt regular intermolecular hydrogen-bonding patterns orreduce backbone complementarity between peptide molecules may decrease self-association while preserving access to nickel-binding functional groups.

[0176]

[0153] In further embodiments, non-fouling behavior may be enhanced by balancing charge distribution and surface properties to reduce non-specific adsorption to biological surfaces or particulate matter present in rumen fluid or manure. Such design considerations may improve functional persistence of nickel-binding peptides by maintaining them in a soluble, bioavailable state capable of sustained nickel sequestration in complex biological matrices.

[0177]

[0154] Peptides exhibiting reduced aggregation or non-specific interactions are expected to maintain higher effective activity in protease-rich and particulate biological environments, thereby improving consistency and durability of nickel-binding function and methanogenesis inhibition under in vivo or environmental conditions.

[0178]

[0155] 5. Methods of Use

[0179]

[0156] 5.1 Inhibiting Methanogenesis in Ruminants

[0180]

[0157] In one aspect, the disclosure provides methods for reducing methane production in a ruminant by administering to the ruminant an effective amount of a nickel chelator (including a protease-resistant nickel-binding peptide), wherein methane production is reduced relative to a control ruminant not receiving the nickel chelator.

[0181]

[0158] Administration routes include, without limitation:

[0182] inclusion in feed as a ruminant feed additive

[0183] inclusion in a total mixed ration (TMR);

[0184] top-dress application;

[0185] delivery as a mineral supplement (e.g. a salt block);

[0186] delivery in drinking water;

[0187] administration as a bolus or slow-release capsule;

[0188] administration via an engineered probiotic which expresses protease-resistant nickel-binding peptides;

[0189] administration via an engineered plant which expresses protease-resistant nickel-binding peptides and / or

[0190] administration via a cannula in research or specialized settings.

[0191]

[0159] In certain embodiments, the nickel chelator is administered at an inclusion rate in feed of about 0.1-100,000 ppm (e.g., 1-10,000 ppm), and / or at about 0.001-100 mg / kg body weight / day(e g., 0.01-10 mg / kg / day), selected to reduce methane while maintaining animal performance. In certain embodiments, animal trace mineral status (including nickel and / or other trace metals) is monitored (e.g., via blood, milk, feces, liver biopsy, or other indicators), and mineral supplementation is adjusted to avoid adverse effects while maintaining local nickel limitation in the rumen.

[0192]

[0160] In certain embodiments, administration reduces methane emissions by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60%, relative to control, as measured by suitable methods of measuring ruminant methane production such as GreenFeed or a respiration chamber. In certain embodiments, nickel chelators are administered during early life stages to influence establishment of the rumen microbiome, resulting in persistent or semi-persistent reductions in methanogenesis even after discontinuation of administration.

[0193]

[0161] In certain embodiments, administration of the composition improves one or more productivity metrics of ruminant agriculture, including, without limitation, increased capture of feed energy as metabolizable energy, improved feed conversion efficiency, increased total volatile fatty acid production, enhanced microbial protein synthesis, increased milk yield or energy-corrected milk yield, increased milk protein content, improved carcass yield or dressing percentage, increased lean tissue accretion, reduced methane emissions per unit of animal product, improved nitrogen utilization efficiency, improved ruminal fermentation stability, reduced time to market weight, and improved reproductive or metabolic health parameters.

[0194]

[0162] 5.2 Inhibiting Methanogenesis in Non-Ruminant Environments

[0195]

[0163] In another aspect, nickel chelators described herein may be applied to inhibit methanogenesis in other environments including, without limitation, wetlands, rice paddies, manure systems, landfills, termite-associated systems, and anaerobic digesters. In these embodiments, nickel chelators may be delivered as powders, granules, liquid formulations, matrices, sorbent materials, plants or microbes engineered to express nickel-binding proteins or peptides or immobilized polymers configured to reduce bioavailable nickel.

[0196]

[0164] In certain embodiments, a nickel chelator is evaluated and / or applied using a controlled microcosm assay comprising: (i) collecting a representative soil / sediment / manure sample, (ii) incubating the sample under anaerobic conditions with a defined organic substrate load, (iii) applying a nickel chelator at one or more dose levels, (iv) measuring headspace methane overtime (e.g., by gas chromatography), and (v) optionally performing a nickel rescue condition by supplementing nickel to confirm nickel-dependent inhibition. Such assays may be used to select chelator dose forms and release profiles suitable for field deployment (e.g., granules, coated particles, slow-release matrices).

[0197]

[0165] 5.3 Modulating Other Nickel -Dependent Biological Processes (e.g., Urease)

[0198]

[0166] In certain embodiments, nickel chelation is used to manipulate nickel-dependent biological processes beyond methanogenesis, including urease-mediated conversion of urea to ammonia. In some embodiments, the disclosure provides methods of increasing a ruminant’s tolerance to nonprotein nitrogen sources (including urea or ammoniated feeds) by administering a nickel chelator, thereby modulating urease activity and avoiding one or more side effects associated with excessive ammonia production.

[0199]

[0167] In certain embodiments, the present disclosure provides methods of modulating one or more nickel-dependent biological processes in a target environment by maintaining bioavailable nickel in the target environment below a level sufficient to support nickel-dependent metabolic activity in methanogenic microorganisms. In some embodiments, such modulation results from limitation of nickel availability rather than from direct inhibition of any individual nickeldependent enzyme.

[0200]

[0168] In certain embodiments, the method further comprises (a) measuring total nickel and / or bioavailable nickel in the target environment; (b) selecting or adjusting an intervention configured to reduce nickel availability based on the measured nickel level; and (c) maintaining bioavailable nickel below a threshold associated with reduced methanogenic activity. In some embodiments, steps (a)-(c) are repeated overtime to dynamically regulate nickel availability in the target environment. In some embodiments, the threshold is selected to reduce methanogenesis while maintaining productivity, fermentation efficiency, or biological function of non-methanogenic organisms.

[0201]

[0169] In certain embodiments, the nickel-dependent biological process comprises methanogenesis. In some embodiments, the nickel-dependent biological process comprises urease-mediated nitrogen metabolism. In some embodiments, the nickel-dependent biological process comprises hydrogen metabolism mediated by one or more nickel-containing hydrogenases.

[0170] In certain embodiments, the target environment comprises a rumen of a ruminant animal. In some embodiments, the target environment comprises an anaerobic digester, manure storage system, agricultural waste collection area, or digestate processing system. In some embodiments, the target environment comprises soil, sediment, wetland, rice paddy, or other anaerobic agricultural or environmental system.

[0202]

[0171] In certain embodiments, bioavailable nickel is reduced by one or more of: (a) sequestration of nickel into a biologically inaccessible form; (b) precipitation or immobilization of nickel; (c) interference with nickel uptake, transport, trafficking, or intracellular delivery; or (d) reduction of dietary or environmental nickel inputs.

[0203]

[0172] 5.4 Inhibiting Methanogenesis and Nitrous Oxide Release in Manure

[0204] In certain embodiments, a nickel chelator administered in vivo is excreted in feces in an active or partially active form and continues to reduce bioavailable nickel in manure following excretion. Such fecal passage embodiments enable restriction of methane production not only within the animal but also during subsequent manure storage, handling, treatment, or land application. In some embodiments, persistence of nickel-chelating activity following excretion is facilitated by structural features of the chelator, including protease resistance, chemical stability, or resistance to degradation under anaerobic or manure-associated conditions.

[0205]

[0173] In related embodiments, nickel chelators are applied directly to manure, slurry, digestate, or litter systems to reduce methanogenesis during storage or treatment. Reduction of bioavailable nickel in such systems limits availability of nickel required for methanogenic metabolism, including activity of nickel-dependent enzymes essential for methane formation, and thereby suppresses methane production in anaerobic manure environments. Such approaches may be applied to liquid or solid manure systems, including storage pits, lagoons, piles, or other manure management structures.

[0206]

[0174] In certain embodiments, nickel chelation in manure systems may also indirectly contribute to reduced emissions of nitrous oxide. Urease is a nickel-dependent enzyme that catalyzes hydrolysis of urea to ammonia and carbon dioxide and plays a central role in nitrogen transformations in manure and agricultural systems. Ammonia generated through urease activity may subsequently undergo microbial nitrification and denitrification processes, which under certain conditions result in formation and release of nitrous oxide. Accordingly, reduction ofbioavailable nickel may limit urease activity and thereby reduce the rate of ammonia generation available for downstream nitrogen oxide-producing pathways.

[0207]

[0175] In anaerobic or intermittently aerobic manure environments, modulation of urease activity and associated ammonia flux may alter microbial community structure and nitrogen cycling dynamics, potentially reducing conditions favorable for nitrous oxide formation. While the extent of such effects may depend on manure composition, microbial populations, oxygen availability, and environmental conditions, limitation of nickel-dependent enzymatic activity provides a mechanistic basis by which nickel chelation may indirectly contribute to reduced nitrous oxide emissions in certain manure management contexts.

[0208]

[0176] 6. Combination Approaches

[0209] Nickel chelation can be combined with other interventions, including:

[0210] a methane inhibitor (e.g., an MCR-targeting compound);

[0211] an acetogenic microorganism and / or acetogenesis-promoting strategy to capture hydrogen that would otherwise be converted to methane;

[0212] a compound or compounds which inhibit growth or viability of methanogens (e.g. lysins) supplementation with one or more non-nickel metals or elements to maintain overall mineral nutrition; and / or

[0213] a masking agent to improve palatability.

[0214]

[0177] In certain embodiments, the nickel chelator is co-administered with 3 -nitrooxy propanol (3-NOP), bromoform or other methane inhibitors, optionally to achieve additive or synergistic methane reductions and / or to reduce dosing requirements.

[0215]

[0178] 7. Formulations and Delivery Systems

[0216]

[0179] 7.1 Masking Agents

[0217]

[0180] In certain embodiments, compositions include a masking agent to increase palatability. Masking agents include sweeteners, flavorings, spices, herbs, and salt mixtures. Non-limiting examples include molasses, corn syrup, honey, vanilla, anise, coriander, clove, ginger, turmeric, and mineral salt mixtures.

[0218]

[0181] 7.2 Slow-Release Capsules and Boluses

[0219]

[0182] In certain embodiments, the nickel chelator is formulated in a slow-release capsule configured to achieve sustained release in the rumen. In certain embodiments, the chelator isformulated in a durable bolus suitable for administration via a balling gun, optionally comprising:

[0220] a dense core to achieve sufficient specific gravity for rumen retention,

[0221] a porous chelator-containing layer (e.g., IIP and / or peptide-containing matrix), and / or an optional outer coating (e g., oils / fats) to facilitate administration.

[0222]

[0183] In certain embodiments, the bolus comprises an RFID or electronic marker to support identification and verification of administration / retention.

[0223]

[0184] 7.3 Microencapsulation and Rumen-Targeted Release

[0224]

[0185] In certain embodiments, nickel chelators (including DMG and / or peptide embodiments) are microencapsulated or coated to improve handling, palatability, and / or controlled release in a target environment. Non-limiting encapsulation / coating strategies include lipid coatings, carbohydrate matrices, pH-responsive polymers, and / or multi-layer coatings configured to release in the rumen while limiting release upstream of the rumen. In certain embodiments, formulations are configured to survive feed processing (e.g., pelleting) and storage while retaining nickel-binding or methane-inhibiting activity upon administration.

[0225]

[0186] 8. Dosing Considerations

[0226]

[0187] Dosing may be selected based on the chelator modality, the target environment, and desired reduction in bioavailable nickel.

[0227]

[0188] In certain embodiments, dosing is selected based on a molar ratio of chelator binding sites to dissolved and / or bioavailable nickel in the target environment, such as at least about 0.5:1, 1:1, 2:1, 5:1, 10:1, or 50:1 (chelating equivalents: estimated bioavailable Ni), optionally accounting for competing metals and matrix effects.

[0228]

[0189] In certain embodiments, DMG may be administered in amounts on the order of at least about 10 mg, 100 mg, 1 g, or 10 g over a defined period (e.g., one month), and / or at doses sufficient to reduce methane production. In peptide embodiments, dosing may be selected to achieve effective nickel sequestration while maintaining stability in the rumen, which may include amounts on the order of at least about 10 mg, at least about 100 mg, at least about 1g or at least about 10g over a defined period (e.g., one day, one week or one month).

[0229]

[0190] Peptides may be administered over time via continuous administration or slow-release formulations to reduce instances of insufficient concentration of the nickel chelator to prevent synthesis of Ni-dependent enzymes.

[0191] 9. Screening and Assays for Nickel -Binding and Functional Inhibition

[0230]

[0192] 9.1 Methanogen Growth Inhibition Assays

[0231] In certain embodiments, candidate nickel chelators and peptides are evaluated using methanogen cultures such as Methanococcus maripaludis. In certain embodiments, growth is monitored by optical density (e.g., ODeoo), which may correlate with methane production, with methane confirmed by headspace gas measurements (e.g., gas chromatography) in follow-on assays.

[0232]

[0193] A nickel chelator candidate may be selected based on one or more criteria including: inhibition of methanogen growth and / or methane production;

[0233] retention of inhibition in the presence of target environment matrix (eg. rumen fluid) or proteases (for peptide candidates); and / or

[0234] reversal of inhibition upon addition of excess nickel (“nickel rescue”), supporting specificity.

[0235]

[0194] 9.2 Protease Challenge and Rumen Fluid Stability

[0236] In certain embodiments, peptide candidates are challenged with one or more proteases (e.g., broad-spectrum proteases) and / or incubated in fresh rumen fluid under anaerobic conditions, and functional activity is assessed following incubation. The Examples include a stability workflow in which peptides are incubated at millimolar concentrations with fresh rumen fluid under anaerobic conditions at physiological temperature and then tested for retained inhibitory function.

[0237]

[0195] 9.3 Metal-Binding Assays and Competition

[0238]

[0196] In certain embodiments, nickel binding is assessed using colorimetric or spectroscopic binding assays, metal competition assays (e.g., Ni vs Zn), precipitation observations, and / or other analytical approaches suitable for evaluating nickel affinity, specificity, and complex stability.

[0239]

[0197] In certain embodiments, functional screening further includes measuring one or more fermentation and microbiome parameters, including volatile fatty acid (VFA) profiles, hydrogen accumulation, acetogenesis markers, and / or relative abundance of methanogens (e.g., via qPCR, sequencing, or metagenomic readouts), to confirm that methane reduction is associated with a desired shift away from methanogenesis rather than general toxicity.

[0240]

[0198] 9.4 Assessment of Nickel-Binding Affinity and Specificity

[0241]

[0199] Nickel -binding affinity of peptide compositions described herein may be determined or inferred using a variety of analytical and functional assays. In certain embodiments, affinity is evaluated using metal-ligand competition assays, including competition with the divalent metal-binding dye 4-(2-pyridylazo)-resorcinol (PAR). PAR forms a colored complex with Ni(II) with a known affinity in the micromolar range, and displacement of Ni(II) from the PAR-Ni complex by addition of a peptide provides evidence that the peptide binds Ni(II) with comparable or greater affinity under the tested conditions. In addition to dye-based competition assays, nickel-binding affinity is further supported by functional biological assays, including nickel-dependent inhibition of methanogen growth and restoration of growth upon supplementation with excess nickel (“nickel rescue”), which demonstrates sequestration of biologically relevant nickel pools. Selectivity for nickel relative to competing metals such as zinc, copper, or cobalt may be assessed by evaluating peptide activity in the presence of these metals at concentrations relevant to the target biological environment. Together, these orthogonal assays provide a reliable basis for inferring nickel-binding affinity and specificity sufficient to reduce bioavailable nickel in biological systems, even where direct thermodynamic dissociation constants are not explicitly measured.

[0242]

[0200] In certain embodiments, nickel-binding affinity is quantified as a dissociation constant (Kd) and may be measured using isothermal titration calorimetry (ITC), spectroscopic titration (e g., UV-Vis or circular dichroism), electrochemical methods, mass-spectrometry-based binding assays, and / or competition assays using indicators or competing ligands having known affinities. In certain embodiments, an apparent Kd is estimated from PAR competition data under defined conditions (e.g., pH, ionic strength) and reported as an apparent Kd for Ni(II) binding in that assay format.

[0243]

[0201] Examples

[0244]

[0202] EXAMPLE 1: Dimethylglyoxime (DMG) inhibits methanogenesis in Methanococcus maripaludis pure culture and rumen fluid cultures

[0245]

[0203] Overview. This Example demonstrates that dimethylglyoxime (DMG), a nickel-selective chelating agent, reduces bioavailable nickel and inhibits methanogen growth and methane production in (i) a defined medium pure culture system using Methanococcus maripaludis and (ii) mixed-media conditions comprising defined medium supplemented with clarified rumen fluid and (iii) diluted rumen fluid cultures. This Example further demonstrates nickel-dependent rescue of growth following growth suppression through DMG, supporting a nickel-specific mechanism of inhibition.

[0204] 1. Materials

[0246] Microorganism. Methanococcus maripaludis (model methanogen).

[0247] Growth medium. McFc defined medium as described by Long et al., 2017 (“A Flexible System for Cultivation of Methanococcus and Other Formate-Utilizing Methanogens,” Archaea, 2017). Chelator. Dimethylglyoxime (DMG), prepared as aqueous stocks at concentrations suitable to achieve test concentrations in culture.

[0248] Nickel Source. Nickel supplied as a soluble nickel salt (e.g., NiCL or NiSO4) for supplementation and rescue conditions.

[0249] Rumen fluid. Clarified, sterilized rumen fluid for hybrid medium experiments, and fresh rumen fluid for diluted rumen fluid culture experiments (source and handling described under Methods below).

[0250] Analytical methods.

[0251]

[0205] Optical density at 600 nm (ODeoo) as a growth proxy.

[0252]

[0206] Gas chromatography (GC) for methane accumulation in culture headspace.

[0253]

[0207] Visual observation / absorbance at -450 nm for Ni-DMG complex precipitation (where applicable).

[0254]

[0208] 2. Methods

[0255]

[0209] 2.1 Anaerobic cultivation conditions

[0256]

[0210] Cultures of AL maripaludis were grown anaerobically at 37°C in sealed glass tubes (e.g., 27.5 mL tubes) comprising (i) a liquid fraction (e.g., -2.5 mL McFc-based medium) and (ii) a headspace (e.g., -25 mL) charged with 20% CO2 and 80% N2 (v / v).

[0257]

[0211] 2.2 Nickel chelation in rumen fluid matrix (visual precipitation)

[0258]

[0212] To demonstrate nickel binding in a biologically relevant matrix, tubes containing a medium comprising -70% clarified rumen fluid were prepared. DMG was added to achieve representative final concentrations (e.g., 0.5 mM or 1.0 mM), and formation of a red insoluble Ni-DMG complex was assessed visually and / or spectrophotometrically (absorbance near 450 nm), see FIG. 1.

[0259]

[0213] 2.3 Dose-dependent growth inhibition in pure culture

[0260]

[0214] AL maripaludis was cultured in McFc basal medium with varying concentrations of DMG (pM range), with or without nickel supplementation (pM range). Growth was monitoredby ODeoo at defined timepoints (e.g., 24-72 h), and compared to a no-DMG, baseline nickel condition, see FIG. 2.

[0261]

[0215] 2.4 Nickel -dependent rescue of M. maripaludis growth (“nickel dependence”)

[0262]

[0216] To assess nickel specificity, cultures inhibited by DMG were supplemented with excess nickel (relative to baseline nickel) and monitored for restoration of growth (ODeoo). The restoration of growth with the addition of nickel suggests that DMG inhibits M. maripaludis growth in a nickel-dependent manner and not through a non-specific toxicity, see FIG. 3.

[0263]

[0217] 2.5 DMG inhibits growth of M. maripaludis in both pure culture and rumen fluid hybrid medium

[0264]

[0218] Headspace methane accumulation was measured by gas chromatography for M. maripaludis cultures grown in: (i) McFc basal medium; and (ii) McFc supplemented with -70% (v / v) sterilized rumen fluid (“hybrid medium”) to simulate the chemical complexity of the rumen environment while retaining the control of a pure culture system. Conditions included nickel supplementation and DMG supplementation, and methane accumulation was normalized to a no-DMG control (100%), see FIG. 4.

[0265]

[0219] This Example supports that nickel-selective chelation is an effective strategy to inhibit methanogenesis. The combination of: (i) visible Ni-DMG complex formation in rumen-fluid matrix, (ii) dose-dependent growth inhibition, (iii) nickel rescue of M. maripaludis growth, and (iv) reduced methane production in both defined and rumen-fluid-containing conditions demonstrates that reducing bioavailable nickel can suppress methanogen growth and methane output.

[0266]

[0220] EXAMPLE 2: Use of Methanococcus maripaludis to screen for methanogen-inhibiting nickel-binding peptides (including nickel specificity and functional stability screening)

[0267]

[0221] Overview

[0268]

[0222] This Example describes a moderately high-throughput screening platform using the model methanogen Methanococcus maripaludis to identify peptide compositions that inhibit methanogen growth (and by extension methanogenesis) via nickel deprivation, and to triage hits based on (i) nickel-dependent rescue (supporting specificity) and (ii) functional stability after incubation in fresh rumen fluid (supporting durability in a relevant biological matrix). This Example further summarizes representative screening outcomes across a large peptide set, provides structure-activity relationship observations derived from HypB-motif variants(including stereochemical constraints relevant to protease resistance), and additionally reports identification of nickel-binding peptides not derived from naturally occurring motifs, including candidates proposed using generative artificial intelligence approaches and validated by the screening assay.

[0269]

[0223] 1. Materials

[0270]

[0224] Microorganism. Methanococcus maripaludis (obligate methanogen).

[0271] Growth medium. McFc medium (e.g., as described by Long et al., 2017).

[0272] Reducing agent. Sodium sulfide (Na2S), added to McFc at a final concentration of about 0.05% (w / v), or as otherwise suitable to maintain anaerobiosis.

[0273] Peptides. Peptides were synthesized by Synpeptide Co., Ltd. (Shanghai, China) using standard solid-phase peptide synthesis methods, supplied as lyophilized powders with vendor-reported purity / identity confirmation (e g., HPLC and mass spectrometry), and stored at approximately -20°C until use.

[0274]

[0225] Peptide handling buffer (“MES-NaCl-DTT”). Sterile buffer comprising 20 mM MES (pH 6), 150 mM NaCl, and 1 mM DTE

[0275]

[0226] Nickel and competitive metals. Nickel supplied as a soluble nickel salt (e.g., NiCL). Where applicable, other divalent metals (e.g., Zn(II), Cu(II)) were supplied as soluble salts for competition / specificity challenges.

[0276]

[0227] Anaerobic atmosphere. 20% CO2 / 80% N2 headspace for cultivation (unless otherwise specified).

[0277]

[0228] 2. Methods

[0278]

[0229] 2.1 General considerations: peptide freshness and trace nickel contamination

[0279]

[0230] Because trace nickel contamination can confound nickel-deprivation assays, reagents and labware were selected and handled to minimize exogenous nickel. Fresh peptide preparations and high-purity reagents were used where possible, and peptide stocks were handled to avoid extended storage or conditions that could compromise activity.

[0280]

[0231] 2.2 Peptide storage and preparation

[0281]

[0232] Lyophilized peptides were stored at approximately -20°C. Peptides were resuspended (inside an anaerobic chamber) to a stock concentration of approximately 10 mM in MES-NaCL DTT buffer. Resuspended peptide stocks were stored at ~4°C for up to approximately one week;activity was observed to become inconsistent and often lower when peptide stocks were stored for significantly longer periods.

[0282]

[0233] 2.3 Nickel background levels in McFc and assay stringency

[0283]

[0234] McFc medium requires addition of nickel (e.g., 1 pM Ni added as NiCh). Independent measurement of total nickel concentration in McFc by atomic absorption spectrophotometry indicated that total nickel may be higher (e.g., -2 pM), consistent with trace nickel contributions from other medium components. This informs assay stringency and was accounted for when selecting peptidemickel ratios and rescue conditions.

[0284]

[0235] 2.4 Culture setup for screening (growth inhibition readout)

[0285]

[0236] A master mix was prepared comprising McFc medium, NazS (final -0.05%), and an actively growing M. maripahidis culture inoculated at approximately 1:1000 (lower inocula are preferred to reduce nickel carry-over introduced with the inoculum). The mixture was distributed into sealed glass tubes (e.g., 2.5 mL liquid in 27.5 mL tubes with -25 mb headspace). Peptides and / or metals were added to achieve desired final concentrations. Control tubes received MES-NaCl-DTT buffer (no peptide). Tubes were sealed and headspace was charged / pressurized with 20% CO2 / 80% N2 (e.g., at -16 psi for at least -30 seconds) and incubated at 37°C.

[0286]

[0237] Growth was monitored by ODsoo at baseline and at one or more timepoints (e.g., 24 h, 48 h, 72 h). Because AT. maripahidis is an obligate methanogen, ODeoo was used as a screening proxy for methane production, with methane measurement by gas chromatography used in follow-on confirmation where appropriate.

[0287]

[0238] 2.5 Screening logic and hit selection criteria

[0288]

[0239] Peptides were selected as nickel-binding methanogen inhibitors that met all three of the following criteria:

[0289]

[0240] Growth inhibition upon peptide addition (consistent with nickel sequestration / limitation);

[0290]

[0241] Nickel-specific rescue: growth is restored toward a no-peptide positive control when the peptide is co-administered with excess nickel, supporting that inhibition is due to nickel limitation rather than nonspecific toxicity; and

[0291]

[0242] Functional stability: growth inhibition is retained after peptide incubation under anaerobic conditions in the presence of fresh rumen fluid for a defined period (e.g., 8-20 hours), indicating resistance to degradation and / or retention of nickel-binding function.

[0292]

[0243] 2.6 Peptidemickel ratio for affinity-focused screening

[0244] To preferentially identify higher-affinity nickel binders, screening was performed using peptide concentrations selected to impose stringent nickel-binding requirements, such as an approximate 10: 1 peptide:nickel molar ratio (e.g., using ~40 pM peptide when total nickel is ~2-4 pM), with the ratio adjustable to tune sensitivity.

[0293]

[0245] 2.7 Nickel rescue condition (specificity testing)

[0294]

[0246] For peptides exhibiting growth inhibition, a rescue condition was established by adding excess nickel (for example, when peptide, which binds nickel in a 1 : 1 molar ratio is tested at -40 pM, nickel may be supplemented to ~50 pM added nickel, or other appropriate rescue levels to exceed the binding capacity of the peptide and ensure adequate nickel) and measuring whether growth returns toward control levels.

[0295]

[0247] 2.8 Functional stability assay in rumen fluid (stability testing)

[0296]

[0248] To evaluate functional stability, peptides were incubated in fresh rumen fluid under anaerobic conditions at ~37°C for up to ~20 hours, and the supernatant was tested for retained inhibitory function in the M. maripaludis assay. In one embodiment:

[0297]

[0249] Fresh rumen fluid is harvested from cows and maintained anaerobically at ~37°C; rumen fluid is used within approximately one hour of collection.

[0298]

[0250] One volume of peptide at -5 mM is mixed with four volumes rumen fluid to yield approximately 1 mM peptide in -80% rumen fluid.

[0299]

[0251] Incubate anaerobically at ~37°C. At timepoints (e.g., T=0 h, T=5 h, T=20 h), centrifuge (e g., 5 min, -14,000 rpm) and filter-sterilize the supernatant (e g., 0.2 pm).

[0300]

[0252] Supplement fdtered supernatant into fresh McFc / Na2S / M maripaludis cultures (e.g., 1:10 v / v) and assess growth inhibition (e.g., ODeoo at -72 h post inoculation).

[0301]

[0253] Ano-peptide rumen fluid control was processed identically.

[0302]

[0254]

[0303]

[0255] 4. Results

[0304]

[0256] 4.1 Summary of screening outcomes

[0305]

[0257] Using the screening system described above, peptides were screened for growth inhibition activity against M. maripaludis often at one or more concentrations and timepoints. In a representative campaign, 44 of 135 peptides tested exhibited “strong inhibition” of at least about 50% under screening conditions (approximately 32% of peptides tested). (FIG. 5) provides representative peptide identifiers / sequences and inhibition categories. For many peptides,inhibition phenotypes were recorded at multiple timepoints (e.g., 24 h, 48 h, 72 h), allowing discrimination between early transient inhibition and sustained inhibition. (FIG. 5) provides representative timepoint-resolved outcomes.

[0306]

[0258] 4.2 Dose dependence, nickel rescue, and competitive metal challenge (representative secondary screening)

[0307]

[0259] Peptides exhibiting inhibitory activity were further evaluated in secondary screening to determine: (1) dose-dependent inhibition; (2) nickel-rescue behavior; and (3) maintenance or loss of inhibition in the presence of competitive metals (e.g., Zn, Cu). (FIG. 6) provides representative results.

[0308]

[0260] In representative embodiments, dose-dependency was observed (increasing peptide concentration correlated with increasing growth inhibition), and nickel-dependent growth restoration was observed upon supplementation with about 50 pM NiCh, supporting nickelspecific mechanisms rather than nonspecific toxicity.

[0309]

[0261] In representative competitive metal testing, peptide SEQ ID NO: 140 retained strong growth inhibition in the presence of ~75 pM Zn, a concentration noted as expected in rumen fluid based on in-house measurements of Zinc in rumen fluid collected from Angus beef cattle at the University of Georgia and measured via ICP-MS. The observation that an excess of Zinc relative to peptide does not fully restore growth as described in FIG. 7 supports the interpretation that the peptide is specific for nickel and nickel binding cannot be abrogated by supplying an excess of a competing metal ion.

[0310]

[0262] 4.3 Functional stability in rumen fluid: L vs D enantiomer performance (representative)

[0263] In a representative embodiment, an all-L peptide (SEQ ID NO: 127; CTTCGCG) and its all-D enantiomer (SEQ ID NO: 140; cttcgcg) were incubated in fresh rumen fluid under anaerobic conditions at 37°C and assayed for retained inhibitory function. The all-L peptide exhibited rapid loss of activity after rumen fluid incubation (partial inhibition at baseline with loss thereafter), whereas the all-D peptide retained inhibitory activity after extended incubation (up to ~20 h), consistent with improved rumen-fluid stability of D-peptides. (Table 2) provides representative results.

[0264] Table 2. Peptide-dependent growth inhibition of M. maripaludis following peptide incubation in fresh rumen fluid. Percent inhibition refers to the level of growth observed as measured by ODeoo relative to controls which did not have peptide added.

[0311]

[0312]

[0265] 4.4 Structure-Activity Relationship evaluation of HypB variants and related motifs

[0266] Background and rationale

[0313]

[0267] A HypB-derived peptide motif was previously described that binds Ni(II) with high affinity and supports square-planar Ni (II) coordination (Chan Chung et al., J. Am. Chem. Soc.

[0314] 2008). For example, a HypB “maquette” peptide (e.g., CTTCGCGEGW) was prepared by solidphase peptide synthesis and shown to bind Ni(II) strongly, with data consistent with a square-planar complex. Chemical modification experiments further suggested that the free N-terminal amine participates as a metal-binding ligand; modification at the N-terminus altered nickel-binding behavior, supporting that the N-terminal amine is part of the nickel coordination sphere together with cysteine ligands.

[0315]

[0268] Consistent with this observation, the inventors found that design operations that reposition, mask, or invert the presentation of the N-terminal amine — such as sequence reversal, retro-enantio conversion, or N-terminal modification — can reduce or abolish nickel-binding activity in certain motifs. Accordingly, in some embodiments, protease resistance is introduced by preparing an analog that retains a free N-terminal amine, or a functional substitute capable of nickel coordination, while converting the motif to a D-amino-acid framework without reversing residue order.

[0316]

[0269] While HypB-derived motifs provide a useful starting point for nickel-binding peptide design, the Chan Chung peptide and related HypB-derived motifs were composed of L-aminoacids and, in the inventors’ studies, L-peptide variants of this motif were found to be rapidly degraded or to lose inhibitory function following incubation in rumen fluid under the conditions described herein (e.g., SEQ ID NO: 127). In contrast, as described above, conversion of the HypB-motif inhibitor sequence to the corresponding D-enantiomer (e.g., SEQ ID NO: 140) yielded a peptide that was substantially more stable in rumen fluid while retaining nickel-binding function and inhibitory activity. Accordingly, while the prior HypB motif demonstrates that short peptides can bind Ni(II), the present results show that effective methanogen inhibition in rumenlike matrices imposes additional, non-trivial constraints relating to peptide stability and stereochemical arrangement.

[0317]

[0270] Structure-Activity Relationship Mapping and Key Observations

[0318]

[0271] Given the remarkable affinity of the HypB motif for nickel, extensive structure-activity relationship mapping was performed for variants of SEQ ID NO: 140 (cttcgcg). The following observations were made:

[0319]

[0272] Threonine correlates with inhibitory activity.

[0320]

[0273] In the tested HypB-motif variants, threonine-containing motifs generally showed stronger inhibition of M. maripaludis growth than related G / C-rich motifs lacking threonine (e.g., SEQ ID NO:117, SEQ ID NOT19, SEQ IDNO:120, SEQ IDNO:121, SEQ IDNO:122, SEQ ID NO:123, SEQ IDNOT24, SEQ IDNO:126, SEQ IDNO:128, SEQ ID NO:129).

[0321]

[0274] Threonine-to-serine substitutions reduce inhibition.

[0322]

[0275] Replacement of threonine residues in SEQ ID NO: 140 with serine (e.g., SEQ ID NO:216) reduced inhibition, consistent with threonine contributing to the active nickel-binding configuration. Without wishing to be bound by theory, the threonine side chain may support a favorable coordination environment.

[0323]

[0276] Non-canonical threonine analog substitutions can preserve inhibition, whereas certain backbone modifications can disrupt inhibition.

[0324] Additional screening demonstrated that substitution of a threonine residue with allo-threonine can preserve strong inhibition in HypB-motif variants (e.g., SEQ ID NO:285 and SEQ ID NO:289). In contrast, substitution with N-methyl-threonine reduced or abolished inhibition in otherwise related sequences (e.g., SEQ ID NO:284 and SEQ ID NO:288). Without wishing to be bound by theory, these results suggest that (i) stereochemical variation at the threonine -carbon (allo-threonine) may be tolerated in the active nickel-binding configuration, whereas (ii)backbone N-methylation at this position may disrupt a backbone-dependent interaction (eg., a required amide N-H and / or local conformational flexibility) important for nickel binding and / or functional nickel deprivation.

[0325]

[0277] C-terminus is relatively permissive but not unlimited.

[0326]

[0278] C-terminal extensions such as 0-Ala linkers and conjugates (e.g., biotin; PEG of various sizes) were generally tolerated (e.g., SEQ ID NO:249, SEQ ID NO:252, SEQ ID NO:256, SEQ ID NO:261, SEQ ID NO:250, SEQ ID NO:254, SEQ ID NO:255). Similarly, incorporation of a,a-disubstituted residues (e.g., a-aminoisobutyric acid (Aib)) as C-terminal extensions was tolerated (e.g., SEQ ID NO:281-283), supporting embodiments in which a,a-disubstituted residues are used to improve protease resistance while preserving inhibitory activity. In contrast to these C-terminal additions which were tolerated, C-terminal proline additions (e.g., SEQ ID NO: 259) reduced activity.

[0327]

[0279] Mixed-chirality variants did not retain inhibition.

[0328]

[0280] Hybrid L / D (mixed-chirality) variants (e.g., SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215) did not retain inhibitory activity, supporting stereochemical constraints on the active configuration.

[0329]

[0281] Reverse-sequence analog is not equivalent.

[0330]

[0282] SEQ ID NO: 140 (cttcgcg) was inhibitory, whereas its reverse-sequence analog SEQ ID NO: 118 (gcgcttc) did not show activity, consistent with sensitivity to residue order and / or terminal group orientation in the nickel-binding configuration.

[0331]

[0283] Collectively, these last two observations regarding chirality support that inhibition depends on the geometric coordination of chelating sites across the peptide, and that improved resistance to degradation via D-amino acids cannot be achieved trivially (e.g., by partial D-substitution or reverso-enantio conversion) without disrupting the nickel-binding configuration required for activity. In certain embodiments, conversion of an L-amino-acid nickel-binding motif to a protease-resistant counterpart is carried out by maintaining the residue order of the parent motif while substituting one or more residues with the corresponding D-enantiomers, including in some embodiments substituting each residue with its D-enantiomer to generate an all-D analog. By contrast, sequence-reversal approaches, including reverse-sequence and / or retro-enantio conversions, may alter the orientation and spacing of coordinating groups and therefore may not preserve nickel-binding activity.

[0284] Non-Limiting Design Considerations

[0332]

[0285] In certain embodiments, a protease-resistant nickel-binding peptide may be derived from a naturally occurring nickel-binding motif by introducing one or more modifications that increase resistance to proteolytic degradation. Such modifications may include, for example, substitution of one or more residues with the corresponding D-enantiomers (including, in some embodiments, substitution of each residue to generate an all-D analog), incorporation of non-canonical amino acids, and / or cyclization.

[0333]

[0286] In these embodiments, retention of nickel-binding activity may be favored by maintaining the spatial presentation of functional groups involved in nickel coordination, such as cysteine thiols and, in some motifs, a terminal amine group. The inventors observed that certain transformations that alter the relative orientation or positioning of such functional groups — such as sequence reversal, retro-enantio conversion, mixed-chirality patterns, or terminal modification — may reduce or abolish nickel-binding activity in some motifs unless nickel-binding activity is retained, as can be assessed using the assays described herein.

[0334]

[0287] The foregoing considerations are illustrative and do not exclude alternative peptide architectures or transformation strategies, provided that nickel-binding function and protease resistance are retained.

[0335]

[0288] 4.5 Discovery of nickel-binding peptides not derived from naturally occurring motifs

[0289] In addition to engineering high-affinity, nickel-selective, and protease-resistant nickel-binding peptides inspired by naturally occurring motifs (e g., HypB-derived motifs), the inventors also identified additional nickel-binding peptide candidates whose sequences are not derived from and are not substantially homologous to such naturally occurring motifs. In certain embodiments, candidate sequences were proposed using computational sequence design methods, including generative artificial intelligence approaches (for example, as described in Zhang et al., 2025 (bioRxiv)).

[0336]

[0290] Using the AL maripaludis screening assay described in this Example, several such non-naturally-derived candidates — including SEQ ID NO:002, SEQ ID NO:264, SEQ ID NO:277, and SEQ ID NO:279 — exhibited strong inhibition at the tested concentration(s). By contrast, a well-known synthetic metal -binding motif, SEQ ID NO:010 (HHHHHH), did not demonstrate inhibition under the screening conditions of this Example.

[0291] These results underscore that functional inhibition in M. maripaludis imposes stringent and non-trivial requirements on nickel sequestration (including affinity, speciation compatibility, and / or stability in the assay matrix), and they further support that effective nickel-binding inhibitors can be discovered through multiple, orthogonal design approaches beyond optimization of known natural motifs, including through the utilization of generative artificial intelligence.

[0337]

[0292] 5. Discussion (Non-limiting)

[0338]

[0293] This Example provides an enabling screening framework for identifying peptide compositions that inhibit methanogen growth by nickel deprivation while prioritizing peptides with nickel specificity (demonstrated by rescue) and durability in rumen fluid (functional stability). The observed stability advantage of D-enantiomer peptides in rumen fluid provides a basis for embodiments incorporating D-amino acids, non-canonical residues, cyclization, and other stability-enhancing modifications while maintaining nickel-binding geometry and specificity. The use of Methanococcus maripaludis as a screening organism in this Example supports applicability beyond ruminant systems. M. maripaludis is an obligate methanogen and serves as a tractable model for archaeal methanogenesis, and methanogenesis pathways (including nickel-dependent processes) are conserved across diverse methanogenic archaea found in environmental and engineered anaerobic habitats (e.g., sediments, wetlands, and anaerobic digesters). Accordingly, nickel-chelating peptides identified using this screening framework may be applied in ruminant and non-ruminant environments to inhibit methanogenesis.

[0339]

[0294] EXAMPLE 3: Peptide-dependent methanogenesis inhibition in fresh rumen fluid cultures

[0340]

[0295] 1. Overview

[0341]

[0296] This Example evaluates whether selected nickel-chelating peptides identified in Example 2 are capable of inhibiting methanogenesis in a mixed-community setting using fresh rumen fluid as the inoculum. In contrast to defined pure-culture assays, fresh rumen fluid contains diverse microorganisms and endogenous substrates; therefore, methane production is quantified directly from tube headspace by gas chromatography rather than through measurement of optical density. The results demonstrate that selected peptides reduce methane formation in diluted rumen fluid cultures relative to no-peptide controls.

[0297] 2. Materials

[0342]

[0298] 2.1 Fresh rumen fluid. Fresh rumen fluid is harvested from beef cattle (e.g., cannulated animals) and maintained under anaerobic conditions at approximately 37°C during transport. The rumen fluid is introduced into an anaerobic chamber and used within less than about one hour following harvest.

[0343]

[0299] 2.2 Growth medium. An anaerobic, sterile growth medium (designated RF-dil medium) previously developed to support the growth of Methanobrevibacter smithii was used (see Kayar et al., Journal of Applied Physiology, 2001, 91(6):2713-2719). Prior to inoculation, the medium was supplemented with sodium sulfide (Na?S) to a final concentration of approximately 0.05% (w / v) to maintain reducing conditions.

[0344]

[0300] 2.3 Test articles. The peptide candidate SEQ ID NO: 140 described in Example 2 is prepared as sterile anaerobic stock solution and added to cultures to achieve the final desired concentration.

[0345]

[0301] 2.4 Culture vessels and gases. Cultures are conducted in sealed glass tubes (e.g., 27.5 mL tubes) containing liquid culture volume and headspace. Headspaces are pressurized using a gas mixture comprising carbon dioxide and hydrogen (e.g., 20% CO2 / 80% FE).

[0346]

[0302] 2.5 Methane analysis. Methane is measured from headspace gas using GC-FID (gas chromatography-flame ionization detection).

[0347]

[0303] 3. Methods

[0348]

[0304] All steps described below are performed in an anaerobic chamber unless otherwise indicated.

[0349]

[0305] 3.1 Preparation of diluted rumen inoculum. Fresh rumen fluid is diluted into RF-DIL (supplemented with Na2S to about 0.05%) to generate a diluted rumen fluid inoculum. In representative embodiments, rumen fluid is diluted at approximately 1:500 (e.g., 0.2 mL rumen fluid into 100 mL medium) or 1 :5000 (e.g., 0.02 mL rumen fluid into 100 mL medium). Lower inocula are generally favored to reduce carry-over of nickel-containing, methane-producing cells; de novo methane production from cells growing in the medium is expected to dominate vs. carry-over from pre-existing cells under these dilute conditions.

[0350]

[0306] 3.2 Aliquoting into tubes and peptide addition. After mixing (e.g., gentle swirling), the RF-DIL-Na2S-rumen fluid (“RF-DIL-Na2S-RF”) culture mix is distributed into glass tubes (e.g., 2.5 mL liquid per 27.5 mL tube, leaving ~25 mL headspace). Peptides are added to thedesired final concentration, with replicate tubes for each condition (e.g., n = 3-5). No-peptide controls contain RF-DIL-NazS-RF culture mix without peptide.

[0351]

[0307] 3.3 Headspace pressurization and incubation. After peptide addition, tubes are sealed, removed from the anaerobic chamber, and headspaces are pressurized with a CO2 / H2 mixture (e g., 20% CO2 / 80% H2) at approximately 30 PSI for at least about 30 seconds. Tubes are incubated at approximately 37°C on slanted racks with gentle shaking (e.g., ~40 rpm) to promote gas exchange (H2).

[0352]

[0308] 3.4 Rationale for methane readout (vs. OD). Optical density at 600 nm (ODeoo) is not recorded in this experiment because there is not a direct correlation between ODeoo and methanogen cell density and / or methane levels in mixed rumen fluid cultures; non-methane-producing microorganisms contribute substantially to optical density in this setting.

[0353]

[0309] 3.5 Headspace flushing and methane measurement. After approximately 24 hours, headspaces are flushed (e.g., by vacuum) and then re-pressurized with H2 / CO2. This step removes methane brought along or produced by the original inoculum, enabling observation of de-novo methane from new cells. After approximately 48 hours, headspace gas samples are collected and methane levels are quantified by GC-FID.

[0354]

[0310] 4. Results

[0355]

[0311] Table 3 summarizes methane production in diluted rumen fluid cultures in the presence of selected peptides. In representative embodiments, methane is reported as % methane vs. nopeptide control at the measurement timepoint (e.g., 48 h). In Table 3, RF means rumen fluid, Avg means Average, SD means standard deviation, N refers to the number of biological replicates included, N’ refers to the number of technical replicates for each biological replicate and Tot refers to the Total number of observations (N x N’)

[0356] Table 3. Effect of selected peptides on fresh RF culture methanogenesis

[0357]

[0358]

[0359]

[0312] 5. Discussion (non-limiting)

[0360]

[0313] This Example extends peptide screening beyond defined pure-culture systems by testing candidate peptides under mixed-community conditions seeded with fresh rumen fluid. The GC-FID headspace methane measurement provides a direct functional readout of methanogenesis in the context of complex rumen microbiota and avoids confounding optical density changes attributable to non-methanogenic organisms present in rumen fluid. The observed reductions in methane relative to no-peptide controls (Table 3) demonstrate that selected peptides can inhibit methanogenesis in diluted rumen fluid cultures, consistent with nickel deprivation as a mechanism for inhibition of methanogen metabolism under complex, environmental conditions and despite the proteolytic environment of the rumen fluid.

[0361]

[0314] In a representative embodiment, SEQ ID NO: 140 (cttcgcg) reduced methane in diluted rumen fluid cultures relative to no-peptide controls, including conditions in which methane decreased from about 16.38 ± 2.63 mM (no-peptide control) to about 2.04 ± 0.44 mM at 10 pM peptide, consistent with substantial functional inhibition in a mixed-community rumen matrix.

[0362]

[0315] EXAMPLE 4

[0363]

[0316] Nickel-binding capacity of peptides using PAR-peptide competition assays

[0364]

[0317] Overview. This Example describes a high-throughput plate-based spectrophotometric competition assay to evaluate Ni(II)-binding by candidate peptides using the colorimetric indicator dye 4-(2-pyridylazo)-resorcinol (PAR). PAR forms a colored complex with Ni(II) that absorbs near 500 nm. When a Ni-binding peptide is present, it competes with PAR for Ni(II), reducing formation of the PAR-Ni complex and decreasing the A500 signal. The assay providesan orthogonal, non-biological measure of peptide nickel binding that can be used to prioritize candidates identified by methanogen inhibition screening (e.g., Example 2) and / or rumen-fluid efficacy tests (e.g., Example 3).

[0365] 1. Materials

[0366]

[0318] Reagents.

[0367] PAR (4-(2-pyridylazo)-resorcinol ) ;

[0368] Ni(II) source (e.g., NiSCE or NiCE);

[0369] Buffer (e.g., HEPES buffer, -50 mM, pH -7.6);

[0370] Candidate peptides (e.g., peptide stocks such as -5 mM in suitable buffer).

[0371]

[0319] Equipment.

[0372] 96-well plate (clear, flat-bottom preferred);

[0373] Plate reader capable of absorbance at -500 nm;

[0374] Multichannel pipettes (optional but recommended);

[0375]

[0320] 2. Methods

[0376]

[0321] 2.1 Assay principle and calculations

[0377]

[0322] PAR binds divalent metals, including Ni(II), and forms a colored complex. In a competition format, peptide-mediated nickel binding is reflected by reduced formation of PAR-Ni complex and therefore reduced absorbance at 500 nm (A500).

[0378]

[0323] For each peptide concentration:

[0379] % Control = A500(PAR + Ni + peptide) / A500(PAR + Ni) x 100;

[0380] % Inhibition = 100 - % Control.

[0381]

[0324] A higher % Inhibition indicates greater peptide competition for Ni(II) under the assay conditions.

[0382]

[0325] Inferring apparent nickel affinity from PAR competition (non-limiting). In some embodiments, the PAR competition assay provides an estimate of an apparent or conditional dissociation constant (Kd,app) for Ni(II) binding by a peptide under the assay conditions (e.g., buffer composition, pH, temperature, and fixed PAR and Ni concentrations). In this context, the peptide concentration that produces 50% inhibition of PAR-Ni signal (“IC50”) is an assaydependent parameter that correlates with relative nickel-binding affinity. Under a competitive binding model in which PAR and peptide compete for the same nickel pool at equilibrium, IC50 may be related to Kd,app for the peptide by a correction term that depends on the concentrationand conditional affinity of PAR for Ni(II). Because PAR-metal complex formation and “effective” stability / dissociation behavior depend on solution conditions and may involve side equilibria, any Kd values inferred from this assay are understood to be approximate and conditional (Kd,app) rather than a directly measured thermodynamic Kd.

[0383]

[0326] In one representative embodiment, SEQ ID NO: 140 exhibits a midpoint of inhibition in the low-tens of micromolar peptide range in the PAR competition format, consistent with a Kd,app for Ni(II) in the micromolar to sub-micromolar range under the tested conditions.

[0384] Accordingly, in non-limiting embodiments, peptides described herein may have a Kd,app for Ni(II) of <50 pM, <25 pM, <10 pM, <5 pM, or <1 pM, as inferred from PAR competition behavior.

[0385]

[0327] 2.2 Representative 96-well plate workflow (non-limiting)

[0386]

[0328] In one embodiment, each peptide is tested across a concentration series generated by serial dilution, with constant final Ni(II) and PAR concentrations.

[0387] 1. Prepare peptide working solution. Prepare a peptide working solution (e.g., 200 pM) by diluting a concentrated peptide stock into HEPES buffer.

[0388] 2. Dispense buffer to plate. Add buffer to wells to support the dilution series and maintain consistent final volume. Include designated no-Ni background wells (PAR + buffer only). 3. Load peptide into row A (highest concentration). Add peptide working solution to row A wells (e.g., in triplicate).

[0389] 4. Serial dilution. Perform serial 2-fold dilutions down the plate to generate a concentration series (e.g., row A highest — row G lowest), leaving row H as 0 peptide controls.

[0390] 5. Add Ni(II). Add Ni(II) solution to all wells except designated no-Ni wells; incubate briefly (e.g., >10 min at room temperature).

[0391] 6. Add PAR. Add PAR solution to all wells to reach final assay concentrations and uniform final volume.

[0392] 7. Read absorbance. Measure A500 and subtract background using the PAR + buffer only wells.

[0393] 8. Compute metrics. Average replicates and compute % Control and % Inhibition.

[0394]

[0329] Non-limiting illustrative final concentrations:Ni(IT): ~10 pM final;

[0395] PAR: -25 pM final.

[0396]

[0330] Peptide: a concentration series (e.g., ~50 pM down to sub-pM range depending on dilution scheme).

[0397]

[0331] Because [PAR] exceeds [Ni] in this illustrative format, the assay reports competition for a limited nickel pool and therefore yields a conditional Kd,app (and / or IC50) that is specific to these solution conditions.

[0398]

[0332] 2.3 Optional variations (non-limiting).

[0399]

[0333] Substitution of other divalent metals (e.g., Co(II), Cu(II), Zn(II)) in place of Ni(II) using the same assay format to assess broader binding behavior and / or selectivity.

[0400]

[0334] Alternative buffers and pH values to evaluate robustness of binding and / or compatibility with physiological matrices.

[0401]

[0335] 3. Results

[0402]

[0336] 3.1 Summary of PAR competition outcomes

[0403]

[0337] Candidate peptides were tested in the PAR-Ni competition assay across a peptide concentration series. Results are reported as A500 values and / or derived % Control and % Inhibition. The complete dataset is provided in FIG. 8.

[0404]

[0338] 3.2 Representative findings and screening thresholds

[0405]

[0339] To identify peptides exhibiting significant nickel competition under the assay conditions, the following non-limiting criteria were applied:

[0406] % Inhibition > 50% at 12.5 pM peptide, and / or

[0407] % Inhibition > 75% at 25 pM peptide

[0408] (under conditions including -10 pM Ni and ~25 pM PAR).

[0409]

[0340] Using these criteria, a subset of peptides met one or more thresholds and were classified as showing significant Ni-binding activity in this assay. In one representative dataset, approximately 36 of 152 peptides (-23%) met at least one criterion.

[0410]

[0341] 4. Discussion (Non-limiting)

[0411] This Example provides an orthogonal, plate-based method for assessing peptide nickel binding that complements biological screening assays. Peptides that strongly inhibit PAR-Ni complex formation under defined conditions are candidates for further evaluation in methanogen inhibition assays, nickel rescue assays, and rumen-fluid stability tests. Conversely, peptides thatshow litle or no PAR competition under these conditions may be deprioritized or further evaluated in alternative binding formats, recognizing that functional inhibition can also depend on nickel speciation, matrix effects, or peptide stability.

[0412]

[0342] EXAMPLE 5

[0413]

[0343] Determination of Acid-Soluble Nickel Concentration in Rumen Fluid (AAS)

[0414]

[0344] Overview.

[0415]

[0345] This Example quantifies acid-soluble nickel concentrations in rumen fluid collected from Angus beef cattle maintained on different diets. The results provide representative baseline rumen nickel concentrations in the low micromolar range, which are relevant to embodiments in which nickel chelation reduces nickel bioavailability to methanogenic microorganisms.

[0416]

[0346] 1. Materials

[0417]

[0347] 1.1 Biological samples.

[0418]

[0348] Rumen fluid samples were collected from Angus beef cattle (n = 18) at the University of Georgia. Animals were maintained on one of the following diet groups:

[0419] Control (grain-fed) (n = 4);

[0420] Low-quality forage (n = 6);

[0421] High-quality forage (n = 8).

[0422]

[0349] Rumen fluid was collected by oral tubing and immediately strained through cheesecloth to remove large particulates and frozen prior to analysis.

[0423]

[0350] 1.2 Reagents.

[0424]

[0351] Trace-metal grade nitric acid (HNCL);

[0425] Atomic absorption-grade nickel (Ni(II)) standard solution.

[0426]

[0352] Trace-metal grade water

[0427]

[0353] 1.3 Equipment.

[0428]

[0354] Graphite furnace atomic absorption spectrophotometer (Shimadzu AA-6701 or equivalent)

[0429]

[0355] 2. Methods

[0430]

[0356] 2.1 Sample preparation and dilution.

[0431]

[0357] Each whole rumen fluid sample was prepared for analysis as follows: frozen rumen fluids were fully thawed in a water bath at 37°C. Then 0.5 mL of rumen fluid was treated (digested)with 20 uL of pure (68-70%) AAS-grade nitric acid HNO3, leading to a final HNO3 concentration of -2.5%. Samples were incubated for at least 12 hours overnight at 60 °C in a water bath. Samples were diluted 1 / 20, 1 / 40 or 1 / 80 as needed, in 1% nitric acid, so that the absorbance values (results) were within the 0-1 pM nickel concentration standard range. Nickel concentrations reported herein were back-calculated to reflect concentrations in undiluted rumen fluid. All values represent acid-soluble nickel measured following acidification and dilution under the conditions described.

[0432]

[0358] 2.2 Standards and calibration.

[0433]

[0359] A nickel standard curve was prepared using an atomic absorption-grade Ni(II) standard diluted in approximately 1% nitric acid. In one embodiment, the calibration range was 0 to 1 pM nickel.

[0434]

[0360] 2.3 Atomic absorption measurement conditions.

[0435]

[0361] Nickel concentrations were quantified by graphite furnace atomic absorption spectroscopy using the nickel calibration curve. A representative sample injection volume of approximately 10 pL was used for each measurement.

[0436]

[0362] 2.4 Replicates and data handling.

[0437]

[0363] Each sample was analyzed in triplicate and up to five replicate measurements were obtained as needed to achieve a coefficient of variation (CV) of <20% across measurements. Where the CV criterion was not met with three replicates, the mean and standard deviation were calculated using all five measurements.

[0438]

[0364] 3. Results

[0439]

[0365] Acid-soluble nickel concentrations in rumen fluid varied among animals and diet groups and were generally observed in the low micromolar range.

[0440]

[0366] Table 4. Acid-soluble nickel concentration in rumen fluids, by diet group.

[0441]

[0442]

[0443]

[0367] 4. Discussion (Non-Limiting)

[0444]

[0368] These results indicate that acid-soluble nickel in strained rumen fluid from Angus beef cattle is typically present at concentrations of approximately 1.5 to 7.1 pM, with an overall mean concentration of approximately 2.84 pM across diet groups. These baseline measurements support embodiments in which reducing rumen nickel bioavailability by chelation shifts nickel concentrations from naturally occurring low-micromolar levels toward lower concentrations expected to suppress nickel-dependent methanogenic metabolism.

[0445]

[0369] Sequence Listing Free Text

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0370] In the sequence cttcgcg-[b-Ala]-[b-Ala]-[PEG8] (SEQ ID NO: 250), PEG8 is -NH-(CH2CH2O)S-H and is covalently attached to the C-terminal carboxyl of [b-Ala] via an amide bond.

[0371] In the sequence cttcgcg-[b-Ala]-[b-Ala]-k-[PEG10K-4Arm] (SEQ ID NO: 254), [PEG10K-4Arm] is a polyethylene glycol polymer with an average molecular weight of 10,000 daltons having 4 arms for attachment to a peptide. The sequence cttcgcg-[b-Ala]-[b-Ala]-k-[PEG1 OK-4 Arm] can be thought of as (cttcgcg-[b-Ala]-[b-Ala]-k)4-[PEGl OK-4 Arm], To synthesize cttcgcg-[b-Ala]-[b-Ala]-k-[PEG10K-4Arm], 4-Arm PEG10K dibenzocyclooctyne (DBCO) of the structure

[0452]

[0453]

[0372] is reacted with four compounds of cttcgcg-[b-Ala]-[b-Ala]-[k(N3)] (SEQ ID NO: 249) using click chemistry (DBCO reacts with the azide of [k(N3)]) to form cttcgcg-[b-Ala]-[b-Ala]-k-[PEG10K-4Arm],

[0454]

[0373] In the sequence cttcgcg-[b-Ala]-[b-Ala]-k-[PEG5K-lArm], [PEG5K-lArm] is a polyethylene glycol polymer with an average molecular weight of 5,000 daltons having 1 arm for attachment to a peptide. To synthesize cttcgcg-[b-Ala]-[b-Ala]-k-[PEG5K-lArm], 1-Arm PEG5K dibenzocyclooctyne (DBCO) of the structure

[0455]

[0456]

[0374] is reacted with cttcgcg-[b-Ala]-[b-Ala]-[k(N3)] (SEQ ID NO: 249) using click chemistry (DBCO reacts with the azide of [k(N3)] to form cttcgcg-[b-Ala]-[b-Ala]-k-[PEG5K-l Arm]).

[0457]

[0375] In the sequence cttcgcggk-(biotin) (SEQ ID NO: 261), the biotin is covalently bonded to the side chain of lysine.

Claims

1. CLAIMS1. A composition comprising a peptide, wherein the peptide(a) binds nickel(II);(b) comprises from 3 to 20 amino acid residues, 3 to 16 amino acids, 3 to 12 amino acids, 3 to 8 amino acids, or 3 to 5 amino acids;(c) comprises at least two sulfur-containing amino acid residues capable of participating in nickel coordination;(d) comprises at least one structural feature that confers increased resistance to proteolytic degradation relative to a corresponding all-L, linear peptide, wherein the structural feature is selected from the group consisting of(i) incorporation of one or more D-amino acid residues;(ii) incorporation of one or more non-canonical amino acids;(iii) incorporation of one or more a,a-disubstituted amino acids;(iv) cyclization of the peptide; and / or(v) incorporation into a cyclotide scaffold; and(e) reduces bioavailable nickel in a protease-rich environment comprising rumen fluid, wherein the reduction in bioavailable nickel is reversible upon supplementation with nickel.

2. The composition of claim 1, wherein the peptide is engineered or non-naturally occurring.

3. The composition of claim 1, wherein each amino acid residue of the peptide is the D-enantiomer of the corresponding amino acid.

4. The composition of claim 1, wherein the peptide comprises one or more non-canonical amino acids selected from -amino acids, N-methylated amino acids, penicillamine, or dehydroalanine.

5. The composition of claim 1, wherein the peptide comprises one or more a,a-disubstituted amino acids that reduce protease susceptibility.

6. The composition of claim 1, wherein the peptide is cyclized by a linkage selected from head-to-tail cyclization, disulfide bonding, lactam formation, thioether formation, or head-to-sidechain cyclization.

7. The composition of claim 1, wherein the peptide comprises a motif of the form Cys-Xn-Cys, wherein n is an integer from 1 to 4 and X is an amino acid residue.

8. The composition of claim 7, wherein at least one X is selected from cysteine, threonine, allo-threonine, serine, valine, a-aminobutyric acid, glycine, alanine, sarcosine, or P-alanine.

9. The composition of claim 7, wherein at least one amino acid residue of the peptide is the D-enantiomer of the corresponding amino acid.

10. The composition of claim 1, wherein the peptide comprises an amino acid sequence selected from the group consisting of the following sequence identities: SEQ ID NO:002, SEQ ID NO: 140 and SEQ IDNO:212.

11. A non-naturally occurring peptide comprising from 4 to 10 amino acid residues, wherein: (a) the peptide comprises a nickel-binding motif of the form Cys-Xn-Cys, wherein n is an integer from 1 to 4 and X is an amino acid residue;(b) the peptide comprises at least one structural feature that contributes to protease resistance selected from:(i) incorporation of one or more D-amino acids;(ii) incorporation of one or more non-canonical amino acids;(iii) incorporation of one or more a, a-di substituted amino acids; and / or(iv) cyclization of the peptide; and(c) the peptide binds nickel(II) and retains functional nickel-binding activity after incubation in rumen fluid at about 37 °C for at least 5 hours.

12. A non-naturally occurring peptide comprising an amino acid sequence of SEQ ID NO:212, wherein:(a) position 2 of SEQ ID NO:212 is an amino acid residue selected from the group consisting of threonine, allo-threonine, serine, valine, and a- aminobutyric acid;(b) position 3 of SEQ ID NO:212 is an amino acid residue selected from the group consisting of threonine, allo-threonine, serine, valine, and a- aminobutyric acid; and(c) position 5 of SEQ ID NO:212 is an amino acid residue selected from the group consisting of glycine, alanine, sarcosine, and P-alanine.

13. The composition of claim 1, wherein the peptide retains functional nickel -binding activity after incubation in rumen fluid at about 37 °C for at least 5 hours.

14. The composition of claim 1, wherein the peptide exhibits an IC50 of <20 pM, or an IC50 of <10 pM, or an IC50 of <5 pM for inhibition of nickel-chelator complex formation in a competitive nickel-binding assay.

15. The composition of claim 14, wherein the peptide binds nickel(II) with an apparent or conditional dissociation constant (Kd,app) of <50 pM, a Kd,app of <25 pM, a Kd,app of <10 pM, a Kd,app of <5 pM or a Kd,app of <1 pM as inferred from competitive binding behavior.

16. The composition of claim 1, wherein reduction of bioavailable nickel by the peptide is sufficient to inhibit methanogenic activity in an anaerobic biological environment17. The composition of claim 1, wherein the peptide is formulated or adapted for administration to a rumen of a ruminant animal.

18. A method of inhibiting methanogenesis in a ruminant animal, comprising administering to the ruminant animal an effective amount of a nickel chelator, wherein the nickel chelator retains nickel-binding activity after exposure to rumen fluid at about 37°C for at least 5 hours, wherein the nickel chelator reduces methane production of the ruminant animal.

19. The method of claim 18, wherein the nickel chelator comprises a peptide of any one of claims 1-17.

20. The method of claim 18, wherein the nickel chelator achieves a reduction in methane production by reducing dissolved or bioavailable nickel to below about 2 micromolar.

21. The method of claim 20, wherein the nickel chelator reduces dissolved or bioavailable nickel to below about 100 nanomolar.

22. The method of claim 18, wherein nickel-dependent methanogenesis inhibition is demonstrated by supplementation with excess nickel to rescue methane production.

23. The method of claim 18, wherein the nickel chelator comprises a peptide comprising a motif of the form Cys-Xn-Cys, wherein n is an integer from 1 to 4 and X is an amino acid residue.

24. The method of claim 18, wherein bioavailable nickel is reduced to a level sufficient to inhibit methanogenesis while systemic depletion of essential trace metals in the host animal is avoided.

25. The method of claim 19, wherein reduction of methane production is achieved at least in part by interference with nickel uptake, transport, trafficking, or intracellular utilization in methanogenic microorganisms.

26. The method of claim 19, wherein reducing bioavailable nickel suppresses methanogenic hydrogen consumption and promotes alternative hydrogen-utilizing microbial pathways in the rumen.

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