Modified plants and plant cells

Abstract

The present invention relates to modified plants, to a method for the production thereof and to a method for reducing methane emissions in animals, in particular ruminant livestock. Plants and plant cells comprising nucleic acid constructs for inducing expression of a polypeptide comprising vanadate dependent halogenated peroxidase (VHPO), or a functional fragment or homolog thereof, are provided.

Description

Technical Field

[0001] The present invention relates to modified plants, methods for their preparation, and methods for reducing methane emissions in animals, particularly ruminant livestock. Background Art

[0002] Ruminants are animals with multiple stomach compartments and include cows, sheep, goats, buffalo, deer, elk, giraffes, and camels. Although ruminants are important for ecological as well as human economic and cultural reasons, such ruminants are also associated with many environmental and economic difficulties.

[0003] First, ruminants emit methane (a potent greenhouse gas) mainly through belching during digestion of food during intestinal fermentation and also through excretion. This contributes to climate change, and it is estimated that 6% of global greenhouse gas emissions are caused by methane emissions from ruminants. Methane is also thought to contribute to the formation of ground-level ozone that is harmful to both humans and the ecosystem, and such ground-level ozone causes lung diseases and deaths, and inhibits and damages plants and crops.

[0004] Second, the feeding efficiency of ruminants is relatively low, in part because ruminants expend energy during belching. This results in ruminants requiring large amounts of land space for growing crops to feed such ruminants and for grazing. Specifically, ruminants require approximately 30% of the earth's habitable land, which places increasing pressure on land use in the context of a growing global population. This can affect the livelihoods of farmers, particularly in developing economies.

[0005] Existing methods for reducing methane emissions and land use requirements in ruminants are limited in terms of scalability, effectiveness, and economic feasibility for farmers globally. Such methods include changing farming systems to improve feeding efficiency and manure management, which are labor- and resource-intensive and require training of farmers, farmer adoption, and implementation consistency; directly modifying the ruminants themselves through breeding and / or genetic modification; and administering vaccine-type treatments to produce antibodies in saliva that inhibit the growth of methanogens in the rumen.

[0006] In Winichayakul et al., “In vitro gas production and rumen fermentation profile of fresh and ensiled genetically modified high–metabolizable energy ryegrass”, Journal of Dairy Science, 2020, perennial ryegrass plants were genetically modified to increase leaf lipid content, resulting in a decrease in the proportion of methane in total gas production. However, the effects on plant vigor and growth are unknown.

[0007] Other methods involve altering the ruminant diet through feed supplements, which may include inhibitor chemical compounds that reduce methane. Some of these methods involve the chemical properties of certain seaweed species.

[0008] Research has shown that when added as a supplement in small amounts, the red seaweed Asparagopsis can significantly reduce methane emissions and improve feeding efficiency (Abbott et al., “Seaweed and Seaweed Bioactives for Mitigation of Enteric Methane: Challenges and Opportunities”, Animals, 2020). WO2021022341A1, WO2018018062A1, WO2020113279A1, and WO2020124167A1 describe the use of Asparagopsis seaweed as an animal feed supplement to reduce methane emissions and enhance immunity. However, it has also been identified that the application of these inventions requires daily feeding of the supplement to ruminants, which is resource–intensive and not practical for many farmers, especially if the ruminants are grazing. In addition, large–scale growth of Asparagopsis seaweed may be difficult and may cause severe damage to the surrounding ecosystem, and the processing and transportation of such seaweed and downstream products may also have environmental impacts.

[0009] WO2020243792A1 discusses genetically modified yeast used as a component of animal feed. The yeast is used to produce bromoform, which is thought to reduce methane production when consumed by ruminants. The application of this invention requires farmers and feed crop suppliers to make an effort to add the yeast to the feed, conduct separate production and quality control of the yeast produced, and monitor the appropriate level of the additive.

[0010] WO2020210074A1 discusses treating pasturelands with beneficial microorganisms and / or their growth by-products to reduce greenhouse gas emissions in one or more ways. WO2019021250A1 describes treating plants or seeds with an algal particle composition. Applying these concepts raises similar questions as discussed above in terms of culturing sufficient amounts of microalgae, the pastureland treatment itself, and the effectiveness of the method.

[0011] Existing methods for reducing the land requirements of ruminants include improving efficiency by managing storage losses, inaccurate feed delivery, animal feed waste, feed nutrient variations, mold, pest damage, and bunk management. Bunk management involves improving the consistency of feed intake by matching the amount of delivered feed to the amount of feed that ruminants can handle. Feed processing is also used, which may involve chopping and processing grains to reduce particle size and improve digestibility, thereby increasing feeding efficiency. Programmed feeding involves a feeding program that achieves a specific weight gain rate while restricting feed intake, which can improve feeding efficiency and reduce fecal production, especially in terms of growth rather than finishing diets. All of these methods require effort from farmers, and using these methods to significantly reduce land requirements is impractical or economically unfeasible for many farmers.

[0012] Therefore, there is a need to provide improved and more effective methods for reducing methane production and other environmental damage in ruminants and for increasing feeding efficiency without the drawbacks associated with existing methods. Summary of the Invention

[0013] In a first aspect, there is provided a nucleic acid construct for inducing the expression of a polypeptide in a plant cell, the nucleic acid construct comprising a coding sequence encoding a polypeptide comprising a vanadate-dependent haloperoxidase (VHPO) or a functional fragment or homolog thereof. The coding sequence is operably linked to one or more regulatory elements adapted to drive the expression of the polypeptide in the plant cell.

[0014] Also provided herein is a modified plant cell comprising a nucleic acid coding sequence encoding a polypeptide comprising a vanadate-dependent haloperoxidase (VHPO) or a functional fragment or homolog thereof. In some embodiments, the modified plant cell comprises the construct described above.

[0015] Also provided herein is a plant comprising the modified plant cell as described. In some embodiments, the plant expresses VHPO in parts edible by livestock (suitably in its leaves and / or grains).

[0016] The plant or the plant cell can produce brominated hydrocarbons, suitably bromoform. The VHPO can be monomeric. In some embodiments, the VHPO is a vanadate-dependent bromoperoxidase (VBPO).

[0017] The nucleic acid of the coding sequence can be DNA, which can be in the form of a plasmid.

[0018] The VHPO polypeptide is derived from a species selected from the group consisting of Asparagopsis spp. (e.g., A. taxiformis, A. armata), Acaryochloris marina, Alaria esculenta, Ascophyllum nodosum, Alteromonas naphthalenivorans; Caulerpa spp. (e.g., Caulerpa taxifolia); Chaetomorpha spp. (e.g., Chaetomorpha linum), Chondrus crispus, Colpomenia sinuosa, Corallina officinalis, Corallina pilulifera, Cystoseira trinodis, Furcellaria spp., Gracilaria spp. (e.g., Gracilaria changii, Gracilaria Gracilariavermiculophylla)); Hormophysa triquetra, Hypnea pannosa, Laminaria spp. (e.g., L. digitata, L. saccharina), Laurencia filiformis, Macrocystis pyrifera, Sargassum flavicans, Zonaria farlowii, Cladophora patentiramea, Dictyota bartayresii, Gigartina spp. (e.g., Gigartina stellata), Oedogonium spp., Padina spp. (e.g., Padina australis), Pterocladia capillacea, and Ulva spp. (e.g., Ulva intestinalis, Ulva linza, Ulva lactuca).

[0019] In some embodiments, the VHPO polypeptide is derived from a microalgal species or a macroalgal species. The microalgal species is a member of the genus Emiliana, Calcidiscus, or Chaetocerus. The macroalgal species is a member of the genus Asparagopsis or Chondrus, and in some embodiments, the VHPO polypeptide is derived from Asparagopsis armata or Asparagopsis taxiformis, and optionally from Asparagopsis taxiformis from the Azores, Asparagopsis armata from the Azores, or Asparagopsis armata from Ireland. In some embodiments, the VBPO is selected from Mbb1, Mbb3, and Mbb4 of Asparagopsis taxiformis.

[0020] In some embodiments, the coding sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NOs: 47, 49 to 54, 57 to 60, 64 to 67, 71 to 74, 77 to 80 and 82 to 87. In some embodiments, the nucleic acid construct, plant cell or plant according to any one of the preceding claims, wherein the coding sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NOs: 47, 49 and 50 (Mbb1, Mbb3 or Mbb4 of Asparagopsis taxiformis). In some embodiments, the coding sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NOs: 54, 57 to 60, 64 to 67, 71 to 74 and 77 to 79 (VHPO of Asparagopsis armata).

[0021] In some embodiments, the construct comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NOs: 52, 58 or 80.

[0022] In some embodiments, the polypeptide comprising the VHPO comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NOs: 1, 3 to 15, 18 to 21, 25 to 28, 32 to 35, 38 to 41 and 43 to 46. In some embodiments, the polypeptide comprising the VHPO comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of Mbb1, Mbb3 and Mbb4 (SEQ ID NOs: 1, 3 and 4) of Asparagopsis taxiformis.

[0023] In some embodiments, the polypeptide comprising the VHPO comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NOs: 15, 18 to 21, 25 to 28, 32 to 35 and 38 to 40 (VHPO of Asparagopsis taxiformis). In some embodiments, the polypeptide comprising the VHPO comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of vBPO of Chondrus crispus (SEQ ID NOs: 5 to 7). In some embodiments, the polypeptide comprising the VHPO comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of the following: vBPO of deep-sea unicellular cyanobacteria (SEQ ID NO: 8); vBPO of Alteromonas naphthalenivorans (SEQ ID NO: 9), vBPO of Ascophyllum nodosum (SEQ ID NO: 10), vBPO of Corallina officinalis (SEQ ID NO: 11), Corallina pilulifera (SEQ ID NO: 12), vBPO of Gracilaria changii (SEQ ID NO: 13) and vBPO of Laminaria digitata (SEQ ID NO: 14).

[0024] The plant cell or the plant is a terrestrial plant. The plant or the plant cell can be a monocotyledonous plant, typically selected from maize, millet, sorghum or forage grass. The plant or the plant cell can be a dicotyledonous plant, suitably selected from alfalfa or clover.

[0025] In some embodiments, the coding sequence is operably linked to a promoter to drive the expression of the polypeptide in a plant cell. The promoter can be a constitutive promoter or an inducible promoter. The promoter can be selected from AlcR / AlcA (ethanol-inducible); GR fusion, GVG and pOp / LhGR (dexamethasone-inducible); XVE / OlexA (β-estradiol-inducible); and heat shock induction.

[0026] In some embodiments, the coding sequence is operably linked to a nucleic acid sequence encoding a targeting signal, and / or the polypeptide can comprise a targeting signal. The targeting signal can be a peroxisome targeting signal, typically wherein the peroxisome targeting signal is SEQ ID NO: 90. The targeting signal can be a mitochondrial targeting signal, suitably wherein the mitochondrial targeting signal is SEQ ID NO: 89. The targeting signal can be a chloroplast targeting signal, typically wherein the chloroplast targeting signal is SEQ ID NO: 88.

[0027] In a second aspect, there is provided an animal feed or animal supplement comprising a modified plant cell or plant of any of the described aspects or embodiments.

[0028] In a third aspect, there is provided a method for reducing methane production in an animal, the method comprising growing or culturing a modified plant or plant cell of any of the described aspects or embodiments under conditions suitable for producing a halogenated hydrocarbon, and feeding an effective amount of the modified plant or plant cell to the animal. An additional method for reducing methane production in an animal, the additional method comprising administering an effective amount of a modified plant cell, plant or animal feed of any of the described aspects or embodiments to the animal.

[0029] In some embodiments, the growth or culturing of the modified plant or plant cell does not include providing a substrate or cofactor associated with the production of a halogenated hydrocarbon. In some embodiments, the halogenated hydrocarbon is bromoform, and the substrate or cofactor associated with the production of a halogenated hydrocarbon includes at least one compound selected from the group consisting of KBr, sodium orthovanadate, pentane-2,4-dione, and H2O2.

[0030] The animal as described may be a ruminant, typically the animal is a bovine. The animal may be a dairy cow, sheep or goat.

[0031] In embodiments of the described method, methane production can be reduced. In some embodiments, the feeding efficiency is improved.

[0032] In a fourth aspect, there is provided a method for preparing a modified plant cell, the method comprising transforming a plant cell with a nucleic acid construct as described in any of the described embodiments. The plant cell may be transformed by Agrobacterium-mediated infiltration. The plant cell may be transformed by particle bombardment.

[0033] In a fifth aspect, there is provided a method for producing an animal feed for reducing methane production in an animal, the method comprising: growing a modified plant cell or plant of any of the described aspects or embodiments; and processing the modified plant cell or plant into an animal feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figures 1A to 1D Exemplary constructs for driving the expression of various genes in maize protoplasts are shown, with relevant reporter genes and targeting sequences where appropriate.

[0035] Figures 2A to 2DShows an exemplary construct for driving the expression of Mbb1 from Asparagopsis taxiformis in Arabidopsis, with associated reporter genes and targeting sequences where appropriate.

[0036] Figures 3A to 3D Shows fluorescence microscopy images of protoplasts transfected with the AtMbb1+GFP fusion construct and the mCherry construct, as detailed in Example 1. Images were taken at 20x magnification in the GFP and mCherry channels with the same acquisition settings and then merged. Figure 3A Shows cells transfected with the cytosolic targeting construct; Figure 3B Shows cells transfected with the chloroplast targeting construct; Figure 3C Shows cells transfected with the mitochondrial targeting construct; Figure 3D Shows cells transfected with the peroxisome targeting construct.

[0037] Figures 4A to 4D Shows fluorescence microscopy images of protoplasts transfected with the CcMbb3+GFP fusion construct and the mCherry construct, as detailed in Example 1. Images were taken at 20x magnification in the GFP and mCherry channels with the same acquisition settings and then merged. 4A shows cells transfected with the cytosolic targeting construct; 4B shows cells transfected with the chloroplast targeting construct; 4C shows cells transfected with the mitochondrial targeting construct; 4D shows cells transfected with the peroxisome targeting construct.

[0038] Figure 5 Shows the morphology of transgenic plant lines 2007, 2011, and 2012 (framed and labeled) and six wild-type Arabidopsis plants (unlabeled), as described in Example 2.

[0039] Figures 6A to 6C Shows the results of transcriptional analysis of transgenic plant lines according to Example 2. At-Mbb1 transcript levels were measured by qRT-PCR and then normalized relative to the expression of a known wild-type gene. Figure 6A Shows the results of Plate 1, also detailed in Table 3. Figure 6B Shows the results of Plate 2, also detailed in Table 4. Figure 6C Shows the results of Plate 1, also detailed in Table 5. Wild-type expression is at the far right of each graph in Figure 6A -C and the normalized expression is zero.

[0040] Figures 7A to 7D Shows the extracted ion chromatogram of transgenic plant material according to Example 5. Figure 7E Is the chromatogram of the positive control: 0.25 μg / mL bromoform standard.Figure 7A It is the chromatogram of the 2007 sample. Figure 7B It is the chromatogram of the 2011 sample. Figure 7C It is the chromatogram of the 2012 sample. Figure 7D It is the gas chromatogram of the 2023 sample.

[0041] Figure 8 It shows the mass spectrometry results of materials taken from the 2007 transgenic plant line, as well as the NIST reference spectrum of bromoform, as detailed in Example 5. The 2007 sample mass spectrometry chart is as shown above, and the NIST reference spectrum is as shown below. Detailed Description of the Invention

[0042] Unless otherwise indicated, the practice of the present invention employs conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA technology, and chemical methods, which are within the capabilities of those of ordinary skill in the art. Such techniques are explained in the following documents, for example, M.R. Green, J. Sambrook, 2012, "Molecular Cloning: A Laboratory Manual", Fourth Edition, Volumes 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel, F.M. et al., ("Current Protocols in Molecular Biology"), John Wiley & Sons, Online ISSN: 1934-3647; B. Roe, J. Crabtree and A. Kahn, 1996, "DNA Isolation and Sequencing: Essential Techniques", John Wiley & Sons; J.M. Polak and James O'D. McGee, 1990, "In Situ Hybridisation: Principles and Practice", Oxford University Press: M.J. Gait (ed.), 1984, "Oligonucleotide Synthesis: A Practical Approach", IRL Press; and D.M.J. Lilley and J.E.Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA, Methods in Enzymology, Academic Press; Synthetic Biology, Part A, Methods in Enzymology, edited by Chris Voigt, Volume 497, pages 2 - 662 (2011); Synthetic Biology, Part B, Computer Aided Design and DNA Assembly, Methods in Enzymology, edited by Christopher Voigt, Volume 498, pages 2 - 500 (2011); RNA Interference, Methods in Enzymology, David R. Engelke and John J. Rossi, Volume 392, pages 1 - 454 (2005). Each of these general texts is incorporated herein by reference.

[0043] Before presenting the invention, a number of definitions are provided that will aid in understanding the invention. All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0044] As used herein, the term "comprising" means that any of the recited elements must be included and other elements may optionally be included. "Consisting essentially of" means that any of the recited elements must be included, elements that would materially affect the basic and novel characteristics of the listed elements are excluded, and other elements may optionally be included. "Consisting of" means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of the present invention.

[0045] "Polynucleotide" is a single-stranded or double-stranded covalently linked nucleotide sequence, wherein the 3' and 5' ends of each nucleotide are linked by a phosphodiester bond. Polynucleotides can be composed of deoxyribonucleotide bases or ribonucleotide bases. Polynucleotides include DNA and RNA, and can be synthesized in vitro or isolated from natural sources. The size of a polynucleotide is usually expressed as the number of base pairs (bp) of a double-stranded polynucleotide, or in the case of a single-stranded polynucleotide, as the number of nucleotides (nt). One thousand bp or nt is equal to a kilobase (kb). Polynucleotides having a length of less than about 40 nucleotides are generally referred to as 'oligonucleotides'. As used herein, the term 'nucleic acid sequence' is a single-stranded or double-stranded covalently linked nucleotide sequence, wherein the 3' and 5' ends of each nucleotide are linked by a phosphodiester bond. Polynucleotides can be composed of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acid sequences can include DNA and RNA, and can be synthesized in vitro or isolated from natural sources.

[0046] Nucleic acids can further include modified DNA or RNA, such as DNA or RNA that has been methylated, or RNA that has undergone post-translational modifications such as 5' capping with 7-methylguanosine, 3' processing such as cleavage and polyadenylation, and splicing. Nucleic acids can also include synthetic nucleic acids (XNAs), such as hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid (LNA), and peptide nucleic acid (PNA).

[0047] According to the present invention, the similarity to the nucleic acid sequences described herein is not limited to 100% sequence identity. In this regard, the term "substantially similar" in relation to two sequences means that the sequences have at least 70%, 80%, 90%, 95%, 98%, 99% or 100% similarity. Similarly, the term "substantially complementary" in relation to two sequences means that the sequences are completely complementary, or at least 70%, 80%, 90%, 95% or 99% of the bases are complementary. That is, mismatches can occur between the bases of the sequences intended to hybridize, and such mismatches can occur between at least 1%, 5%, 10%, 20% or up to 30% of the bases.

[0048] When applied to a nucleic acid sequence, for example in an expression construct, the term 'operably linked' indicates that the sequences are arranged so that they act in concert to achieve their intended purpose. For example, in a DNA vector, a promoter sequence permits initiation of transcription through an adjoining coding sequence up to a termination sequence. In the case of an RNA sequence, one or more untranslated regions (UTRs) may be arranged relative to an adjoining polypeptide coding sequence referred to as an open reading frame (ORF). A given mRNA as disclosed herein may contain more than one ORF, i.e., a so-called polycistronic RNA. As is known in the art, an mRNA may encode more than one polypeptide and may thus include cleavage sites or other sequences required to produce multiple functional products. A UTR may be located 5' or 3' relative to an ORF of a coding sequence to which it is operably linked. A UTR may contain sequences found in mRNA sequences typically found in nature, such as any one or more of the following: Kozak consensus sequences, start codons, cis-acting translational regulatory elements, cap-independent translation initiator sequences, poly-A tails, internal ribosome entry sites (IRESs), structures that regulate mRNA stability and / or lifetime, sequences that direct mRNA localization, and the like.

[0049] In the context of the present invention, the term 'expressing a polypeptide' means producing the polypeptide encoded by the polynucleotide sequence described herein. Generally, this involves transcription of a DNA sequence contained within a modified plant cell, followed by translation of the resulting mRNA sequence by the ribosomal machinery of the plant cell containing the sequence.

[0050] As used herein, the term 'polypeptide' is a polymer of amino acid residues linked by peptide bonds, whether produced naturally or in vitro by synthetic means. Polypeptides having a length of less than about 12 amino acid residues are generally referred to as "peptides", and polypeptides having a length between about 12 and about 30 amino acid residues may be referred to as "oligopeptides". The term "polypeptide" as used herein denotes naturally occurring polypeptides, precursor forms or products of pre-proteins. Polypeptides may also be subject to maturation or post-translational modification processes, which may include, but are not limited to: glycosylation, proteolytic cleavage, lipidation, signal peptide cleavage, propeptide cleavage, phosphorylation, etc. The term "protein" is used herein to refer to macromolecules that comprise one or more polypeptide chains. Some proteins are multimeric or involve complexes of polypeptide chains and other molecules (such as cofactors). In such cases, the polynucleotide encoding such a protein as described herein is intended to cover the provision of multiple monomers such that the complete or functional resulting protein can be produced. The different monomers may be encoded on the same polynucleotide construct or separately encoded. Transcription / translation of the modified intracellular polynucleotide sequence allows for the imposition of local post-translational modifications appropriate for the cell type. Such modifications may regulate the folding, localization, interaction, degradation and activity of the gene product. Typical post-translational modifications may include cleavage, refolding and / or chemical modifications such as methylation, acetylation or glycosylation.

[0051] A 'promoter' is defined as an array of nucleic acid control sequences that direct the transcription of an operably linked nucleic acid. The promoter includes the essential nucleic acid sequences near the transcription start site, such as the TATA element in the case of a polymerase II type promoter. The promoter also optionally includes distal enhancer or repressor elements, which may be located up to several thousand base pairs away from the transcription start site.

[0052] The present invention seeks to alleviate the disadvantages of existing methods for reducing methane emissions and land requirements of ruminants by providing a solution that is globally scalable, economically beneficial to farmers and has the potential to continuously reduce methane emissions and improve feeding efficiency (and reduce the overall land requirement for ruminants).

[0053] Methane is a greenhouse gas that can be produced by methanogenic microorganisms, particularly methanogenic archaea. Without wishing to be bound by theory, it is thought that these microorganisms exist not only in the environment but also inhabit the gastrointestinal tract of ruminants, thereby contributing to methane production. It is also thought that the presence of halogenated hydrocarbons (such as brominated hydrocarbons) can reduce methane production in this context. Brominated hydrocarbons are thought to prevent methane emissions from ruminants by interfering with the vitamin B12 cofactor, which is used by the methyltransferase of ruminant methanogenic archaea to produce methane.

[0054] As mentioned, various macroalgae have been shown to reduce methane emissions when fed to ruminants, with the red macroalga Asparagopsis shown to be highly effective. These species produce halogenated compounds, including bromoform (CHBr3) and polybromomethanes (such as dibromomethane and chlorodibromomethane), which are thought to be synthesized by the catalytic conversion of halide anions to hypohalous acids by haloperoxidases, where hydrogen peroxide (H2O2) acts as an oxidant. The requirement for H2O2 links the synthesis to the enzymatic production of reactive oxygen species (ROS).

[0055] "The gene cluster containing the enzymes that produce reactive oxygen species (ROS) and the vanadium-dependent haloperoxidase (VHPO) that produces bromoform in Asparagopsis taxiformis" was identified in Thapa et al., "Genetic and biochemical reconstitution of bromoform biosynthesis in Asparagopsis lends insights into seaweed ROS enzymology" (ACS Chem Biol, 2020). It was identified that Mbb1, Mbb3, and Mbb4 VHPOs could synthesize bromoform in in vitro assays when provided with sodium orthovanadate, potassium bromide, H2O2, and the artificial substrate monochlorodimedone (MCD) to the recombinant proteins expressed in Escherichia coli (E. coli), presenting the following equation. After testing a series of hydrocarbon substrates, it was also proposed that acetoacetyl-S-ACP (an acyl-acyl carrier protein intermediate in plastid fatty acid synthesis) might be the physiological substrate for bromoform synthesis in A. taxiformis. The authors of Thapa et al. were unable to heterologously express the putative ROS-producing enzyme Mbb2, but did identify the Chondrus crispus homolog and demonstrate its NAD(P)H-oxidase (NOX) activity. They also demonstrated the activity of the homologous C. crispus VHPO protein.

[0056]

[0057] A more general alternative equation for the production of brominated organic compounds is

[0058] RH + HBr + H2O2 → RBr + 2H2O

[0059] where RH is an organic substrate.

[0060] VHPO enzyme sources and uses

[0061] In embodiments of the present invention, a plant or plant cell is modified to express a substance that reduces methane production in an animal, typically a ruminant, that ingests the plant, plant cell, or feed prepared therefrom. In this context, a plant is a member of the plant kingdom, typically its terrestrial members. Sources of genetic modification of such plants are often found in aquatic photosynthetic species such as algal species and phytoplankton, several of which produce substances that reduce methane production in animals. It is contemplated to introduce the genetic basis for the production of these substances into plants, particularly ruminant forage plants (forage grasses), to effect efficient uptake of appropriate amounts of these substances. Thus, the plants or plant cells of the present invention can be modified with a construct comprising a coding sequence (e.g., a coding sequence encoding VHPO) that is derived from an aquatic photosynthetic species such as a microalgal species or a macroalgal species. In this context, 'derived from' means that the construct encodes a polypeptide that is functionally and / or sequentially similar to a polypeptide produced by a wild-type member of these species. Such constructs can differ from the original construct by including codon optimization, the presence of reporter gene sequences, targeting sequences, modified nucleobases, etc. It is contemplated that VHPO enzymes and other proteins as described herein can be engineered or modified by any of a number of methods known in the art to increase their activity, stability, or other properties.

[0062] In some embodiments, the present invention provides plants and plant cells that are modified to express VHPO and / or otherwise produce a halogenated hydrocarbon such as bromoform to reduce methane production in an animal. Similarly, suitable VHPO enzymes are present in many aquatic photosynthetic species. A VHPO enzyme specialized for the production of a halogenated hydrocarbon can be referred to as a vanadium-dependent bromoperoxidase (VBPO), and VHPO as referred to herein is intended to encompass VBPO. It is expected that the presence of VHPO alone is sufficient to produce a halogenated hydrocarbon by the modified plants and plant cells of the present invention.

[0063] Species that have been identified as expressing VHPO and / or having methane-reducing effects include Asparagopsis (e.g., Asparagopsis taxiformis, Asparagopsis armata), deep-sea unicellular cyanobacteria, Alaria esculenta, Ascophyllum nodosum, Alteromonas naphthalenivorans; Caulerpa (e.g., Caulerpa taxifolia); Cladophora (e.g., Cladophora linearis), Chondrus crispus, Cystoseira, Corallina officinalis, Jania rubens, Dictyota dichotoma, Gracilaria (e.g., Gracilaria changii, Gracilaria vermiculophylla); Sphaerotrichia divaricata, Champia parvula, Laminaria (e.g., Laminaria digitata, Saccharina japonica), Laurencia filamentosa, Macrocystis pyrifera, Sargassum, Padina faulkneri, Cladophora bombycina, Dictyota bartayresiana, Gelidium (e.g., Gelidium divaricatum); Oedogonium, Padina (e.g., Padina australis), Ulva (e.g., Enteromorpha intestinalis, Enteromorpha linza, Ulva lactuca) (see, e.g., Thapa et al., 2020, WO 2020 / 243792, Abbott et al., 2020, Thapa, H.R. and Agarwal, V. (2021)). Obligate brominating enzymes underlie bromoform production by marine cyanobacteria (Obligate Brominating Enzymes Underlie Bromoform Production by Marine Cyanobacteria). Journal of Phycology). Microalgal species identified as expressing VHPO include members of the genera Emiliania, Calcidiscus, and Chaetoceros. Thus, nucleic acid sequences encoding polypeptides (such as VHPO from any of these species) can be used in this context. VHPO can be monomeric or not monomeric.

[0064] Compared to Asparagopsis armata, which is adapted to colder temperate waters, Asparagopsis taxiformis preferentially grows in tropical to warm temperate waters, but the species overlap in some environments, such as the Mediterranean (Andreakis et al. (2004) “Asparagopsis taxiformis and Asparagopsis armata (Bonnemaisoniales, Rhodophyta): genetic and morphological identification of Mediterranean populations” European Journal of Phycology). In fact, each subspecies or strain is found in different locations, for example, Asparagopsis taxiformis has Atlantic and Mediterranean strains, while Asparagopsis armata has been isolated near Ireland and the Azores.

[0065] To identify additional potential VHPOs for use in the current context, genomic comparisons were made of the identified Mbb1, Mbb2, Mbb3, and Mbb4 polypeptides identified in Asparagopsis taxiformis (SEQ ID NOs: 1 to 4) and Chondrus crispus (SEQ ID NOs: 5 to 7) with related species Asparagopsis armata isolated from strains in Ireland (Asparagopsis armata from Ireland) and the Azores (Asparagopsis armata from the Azores), and an Asparagopsis taxiformis strain isolated from the Azores (Asparagopsis taxiformis from the Azores).

[0066] Multiple collections of the macroalgae were made and DNA and RNA preparations were generated for sequence analysis using Illumina sequencing technology. Library preparation for genomic DNA sequencing included DNA fragmentation, adapter ligation, size selection, and amplification. It included unique dual indexing. The run type was paired-end, where the read length was 2 x 150 bp. Over 5 million read pairs (10 million reads) (+ / - 3%) were obtained per sample; additionally, transcriptome sequencing was completed in triplicate for each sample in the samples using Illumina sequencing technology. Library preparation for RNA sequence analysis included strand-specific cDNA library preparation, purification of poly-A containing mRNA molecules, mRNA fragmentation, randomly primed cDNA synthesis (strand-specific), adapter ligation, and adapter-specific PCR amplification. The run type was paired-end, where the read length was 2 x 150 bp. 30 million read pairs (+ / - 3%) were identified. The method included long and accurate read lengths for quantitative assessment of common and rare transcripts. The genomic DNA and transcriptome sequencing data were mapped against taxonomically distinct reference genomic sequences and otherwise analyzed for identification and annotation of gene coding sequences, identification and quantification of transcripts, pairwise comparison of expression levels and determination of significant fold differences, alternative splicing analysis based on known / provided eukaryotic gene models, and detection and annotation of SNPs and InDels. The transcriptome sequencing data also provided comprehensive information on gene expression, levels, sequences, structures, and strand orientations of RNA types. BLASTN searches were used to search the coding sequences identified in this analysis to identify sequences related to Mbb1, 2, 3, and 4. This analysis enabled the identification of Mbb1, 2, 3, and 4 coding sequences in Asparagopsis taxiformis from the Azores and Asparagopsis armata from Ireland and the Azores. Additionally, two Asparagopsis armata strains were found to have a second Mbb4 gene not present in Asparagopsis taxiformis. The identified polypeptides and nucleotide coding sequences are shown in Table 6.

[0067] Thus, the plants or plant cells described herein can be modified to express one or more proteins identified in *Asparagopsis taxiformis* and / or *Asparagopsis armata*, suitably the VHPO identified in one or both of these species. In some cases, the plants or plant cells are modified to express *Asparagopsis armata* proteins selected from those identified as Mbb1-like, Mbb3-like, Mbb4-like and Mbb4-2-like, and specifically the Mbb4-2-like protein.

[0068] Inducible expression or activity of VHPO

[0069] In some cases, it may be advantageous to control the degree to which VHPO is expressed by the modified plant cells or plants and / or the activity of the VHPO polypeptide synthesized by the plants or plant cells. Halogenated hydrocarbons can have toxic effects on the organisms in which they are expressed and / or on the environment. For example, halogenated hydrocarbons are thought to play a role in ozone depletion.

[0070] In addition, depending on the cell type and / or the location of the cell within the plant, it may be beneficial to control the expression of the encoded VHPO. For example, expressing VHPO only in the parts of the plant (such as leaves or grains) that are eaten by a particular animal may be advantageous for efficiency. This can also limit any toxic effects of the production of VHPO or subsequently produced halogenated hydrocarbons.

[0071] It is contemplated that the desired compounds (such as bromoform) are produced in any plant tissue that is typically fed to ruminant livestock. This can include but is not limited to leaves and stems (e.g., from forage / cereals, legumes and brassicas), seeds (e.g., from cereals, legumes and oilseed crops) and roots / tubers (e.g., from turnips, swedes, sugar beets).

[0072] Plant gene promoters, including constitutive promoters; plant gene promoters for genes having tissue-specific or cell-specific or tissue-enhanced or cell-enhanced expression; and plant gene promoters that are regulated in response to environmental, hormonal, chemical and / or developmental signals, such as those discussed in US 9,556,102 B2, which is incorporated herein by reference.

[0073] Accordingly, it is envisioned that the nucleic acid coding sequences described herein are operably linked to one or more promoters or other control sequences that allow control of the expression of VHPO, such as its transcription, translation, or otherwise. Such promoters are also referred to as "inducible promoters" as compared to "constitutive promoters" that are active in all cases. In some cases, inducible promoters only allow expression in the presence or absence of specific conditions, such as temperature, humidity, or the presence of one or more chemical signals. For example, inducible transgene expression that can be used in modified plant cells is discussed in Borghi "Inducible gene expression systems for plants". Methods Mol Biol. 2010, and Misra and Ganesan "The impact of inducible promoters in transgenic plant production and crop improvement" Plant Gene, 2021, and includes AlcR / AlcA (ethanol inducible); GR fusions, GVG, and pOp / LhGR (dexamethasone inducible); XVE / OlexA (β-estradiol inducible); and heat shock induction.

[0074] Other methods of controlling the expression of the provided polynucleotide coding sequences include using post-transcriptional control mechanisms. For example, the provided coding sequences can be operably linked to microRNA sequences, allowing for downregulation of expression in specific cell and / or tissue types and subsequent expression in other cell and / or tissue types.

[0075] Additional methods of controlling the activity of the expressed VHPO include expressing the polypeptide in an inactive peptide form and then activating the polypeptide in a particular situation. Alternatively, the substrate (such as a carbon source), cofactor, and / or other compounds or conditions required for the VHPO to function can be withheld until halogenated hydrocarbon production is desired. In some embodiments, the modified plant cell or plant can be grown in the presence of vanadium or a vanadium-containing compound (such as sodium orthovanadate) only when halogenated hydrocarbon production is desired. Similarly, the modified plant cell or plant can be grown in the presence of a halogen or halide (suitably bromine or a bromide such as sodium bromide or potassium bromide) only when halogenated hydrocarbon production is desired. In some embodiments, the modified plant or plant cell can produce or provide or be modified to further produce or provide the substrate (such as a hydrocarbon source), cofactor, and / or other compounds or conditions required for the VHPO to function. In such cases, the production of these secondary factors can alternatively or additionally be controlled by any suitable means, such as the means described above.

[0076] In such cases, some embodiments of the methods described herein may involve providing the necessary inducer, substrate, or cofactor as indicated above only when VHPO expression and / or halogenated hydrocarbon production is desired. Similarly, when VHPO expression and / or halogenated hydrocarbon production is not desired, the inducer, substrate, or cofactor can be depleted or removed. For example, the vanadium compound can be chelated to eliminate its availability.

[0077] It is contemplated that bromoform production can be induced in the modified plants or plant cells as described herein by subjecting the plants or plant cells to one or more stressors (e.g., heat stress or drought stress). Without being bound by theory, it is believed that the level of reactive oxygen species (ROS) can increase in stressed plants and can thus be more available for the VHPO described herein.

[0078] As described above, the modified plants or plant cells can be modified to further produce or provide the substrate (such as a hydrocarbon source), cofactor, and / or other compounds or conditions required for the VHPO to function. For example, the plant or plant cell can be modified to produce additional ROS, such as by modifying to produce a protein that produces or increases the production of ROS. Such a protein can be a NADPH oxidase, such as the Mbb2 NADPH oxidase from Asparagopsis taxiformis or its equivalents or homologs. Conversely, when modification is desired only to produce VHPO, the plant or plant cell can not be modified to produce NADPH oxidase (such as Mbb2 and identified equivalents). Such modification can be carried out and controlled by any suitable means, such as the means described herein.

[0079] Organelle-targeted expression

[0080] Since the VHPO protein is a peroxidase, it therefore requires H2O2 and may require a carbon source to function. The availability of these substrates varies within the cell and, specifically, may be more abundant in peroxisomes, chloroplasts, and / or mitochondria, as these organelles are particularly involved in the production and / or removal of reactive oxygen species. Thus, by targeting a protein, such as VHPO, to a subcellular location, the present invention can reduce or eliminate the need for additional substrates and / or cofactors associated with the production of halogenated hydrocarbons (e.g., bromoform) to be provided to a plant or plant cell.

[0081] Additionally, targeting a protein, such as VHPO, to a subcellular location can be used to protect the cell from any harmful or toxic effects of any product, such as bromoform.

[0082] Thus, the polypeptides and / or encoding nucleic acid sequences of the present invention can comprise or encode targeting sequences adapted to target a specific cellular location, suitably targeting peroxisomes, chloroplasts, and / or mitochondria. The targeting sequences can be selected to match the sequences used by the species to be modified or, where appropriate, can be selected from another species. The targeting sequence can be located at the C-terminus, N-terminus, or elsewhere on the polypeptide.

[0083] Modifying plant cells and plants for use

[0084] The present invention is intended to encompass the modification of any suitable plant or plant cell, which is typically a terrestrial plant or its cell suitable for feeding to a ruminant. However, in some embodiments, aquatic plants can be used. The skilled person will be able to select suitable candidates for modification depending on the animal, environment, climate, and / or the amount required to achieve methane reduction.

[0085] In the context of the present invention, reference to a plant cell herein is intended to encompass a plant protoplast, i.e., a plant cell lacking a cell wall, or a plant cell that has been treated to remove the cell wall. When in the protoplast state, the plant cell is typically modified. It is envisaged that the modified plant cells according to the present invention can be cultured in vitro, as protoplasts or in other forms, and fed to animals in a processed or unprocessed form, i.e., in a process that does not regenerate the whole plant. Similar methods are envisaged using callus cells or other plant cells growing in suspension.

[0086] The plant or plant cell can be a monocotyledon or a dicotyledon. Suitable monocotyledons include maize, millet, sorghum, and forage grasses. Suitable dicotyledons include alfalfa and clover.

[0087] Forage grasses that can be used in the current context include Agrostis spp. (or bentgrasses), Agrostis capillaris (or common bentgrass), Agrostis stolonifera (or creeping bentgrass), Andropogon hallii (or sand bluestem), Arrhenatherum elatius (or false oat-grass), Bothriochloa bladhii (or Australian bluestem), Bothriochloa pertusa (or hurricane grass), Brachiaria decumbens (or Surinam grass), Brachiaria humidicola (or koroniviagrass), Bromus spp. (or bromegrasses), Cenchrus ciliaris (or buffelgrass), Chloris gayana (or Rhodes grass), Cynodon dactylon (or bermudagrass), Dactylis glomerata (or orchard grass), Echinochloa pyramidalis (or antelope grass), Entolasia imbricata (or bungoma grass), Festuca spp. (or fescues), Festuca arundinacea (or tall fescue), Festuca pratensis (or meadow fescue), Festuca rubra (or red fescue), Festulolium (or Festuca-Lolium hybrids), Heteropogon contortus (or blackspear grass), Hymenachne amplexicaulis (or West Indian marsh grass), Hyparrhenia rufa (or jaragua), Leersia hexandra (or southern cutgrass), Lolium spp.Or ryegrasses), Italian ryegrass (Lolium multiflorum), perennial ryegrass (Lolium perenne), Guinea grass (Megathyrsus maximus), molasses grass (Melinis minutiflora), carabao grass (Paspalum conjugatum), dallisgrass (Paspalum dilatatum), reed canarygrass (Phalaris arundinacea), timothy (Phleum pratense), bluegrasses and meadow - grasses (Poa spp.), Texas bluegrass (Poa arachnifera), Kentucky bluegrass (Poa pratensis), rough bluegrass (Poa trivialis), African bristlegrass (Setaria sphacelata), kangaroo grass (Themeda triandra), intermediate wheatgrass (Thinopyrum intermedium).

[0088] Beans that can be used in the current context include pinto beans (Arachis pintoi or pinto peanut), cicer milkvetch (Astragalus cicer or cicer milkvetch), roundleaf sensitive pea (Chamaecrista rotundifolia or roundleaf sensitive pea), butterfly-pea (Clitoria ternatea or butterfly-pea), annual lespedezas (Kummerowia or annual lespedezas), Korean clover (Kummerowia stipulacea or Korean clover or Korean lespedeza), Japanese clover and common lespedeza (Kummerowia striata or Japanese clover and common lespedeza), bird's-foot trefoil (Lotus corniculatus or bird's-foot trefoil), purple bush-bean (Macroptilium atropurpureum or purple bush-bean), burgundy bean (Macroptilium bracteatum or burgundy bean), medics (Medicago spp. or medics), alfalfa or lucerne (Medicago sativa or alfalfa or lucerne), barrel medic (Medicago truncatula or barrel medic), sweetclovers (Melilotus spp. or sweetclovers), perennial soybean (Neonotonia wightii or perennial soybean), common sainfoin (Onobrychis viciifolia or common sainfoin), stylo (Stylosanthes spp. or stylo), Townsville stylo (Stylosanthes humilis or Townsville stylo), shrubby stylo (Stylosanthes scabra or shrubby stylo), clovers (Trifolium spp. or clovers), alsike clover (Trifolium hybridum or alsike clover), crimson clover (Trifolium incarnatum or crimson clover), red clover (Trifolium pratense or red clover), white clover (Trifolium repens or white clover), broad beans (Vicia spp.or vetches), Vicia articulata or oneflower vetch, Vicia ervilia or bitter vetch, Vicia narbonensis or narbon vetch, Vicia sativa or common vetch or tare, Vicia villosa or hairy vetch, Vigna parkeri or creeping vigna.

[0089] Cereals that can be used in the current context include Zea mays or maize or corn, Triticum spp., Triticum aestivum or bread wheat, Oryza sativa or Asian rice, Oryza glaberrima or African rice, Hordeum vulgare or barley, Avena sativa or oats, Secale cereale or rye, Sorghum spp., Sorghum bicolor, millet (e.g., Panicum miliaceum, Panicum sumatrense, Panicum sonorum, Pennisetum glaucum).

[0090] Root crops that can be used in the current context include Raphanus sativus or Daikon or radish, Brassica rapa or turnip, Brassica napus or Rutabaga or Swede or forage rape, Beta vulgaris or sugar beet.

[0091] Pulses and oilseeds that can be used in the current context include soybean (Glycine max or Soybean), common bean (Phaseolus spp.), kidney bean (Phaseolus vulgaris or Kidney bean or navy bean or pinto bean or black turtle bean or haricot bean), broad bean (Vicia faba or broad bean or field bean), pea (Pisum sativum or garden pea), white lupin (Lupinus albus or lupin), oilseed rape or canola, sunflower (Helianthus annuus or sunflower), peanut (Arachis hypogaea or peanut or groundnut).

[0092] In addition to feed plants for direct feeding to ruminants, it is contemplated that plants and plant cells of other species can be transformed as discussed herein. For example, model plant species can be used, such as Nicotiana benthamiana, Arabidopsis thaliana (both eudicotyledons); and the Lemnoideae (including Lemna spp.) or rice (monocotyledon).

[0093] Any suitable method can be used for transient and / or stable transformation of plant cells, as will be obvious to those skilled in the art in the context. For example, Agrobacterium-mediated transformation, chemical procedures, electroporation, and / or use of high-velocity particles (particle bombardment) can be used. Genome editing techniques known in the art can also be used. For example, methods for plant transformation, plastid transformation, and regeneration, development, and cultivation of plants from single plant protoplast transformants are discussed in US 9,556,102 B2 (incorporated herein by reference).

[0094] Similarly, the nucleic acid constructs of the present invention or nucleic acid constructs consisting of the plants or plant cells of the present invention can be in any suitable form. DNA or RNA sequences can be used. Where short-term or transient production is preferably appropriate, mRNA sequences can be used because the provided mRNA represents an expendable supply of the genetic code for producing a specific polypeptide. Circular RNA can also be used. The DNA sequence can likewise be in any suitable form, such as a plasmid. The nucleic acid construct and / or transformation method can be selected for stable transformation, such as by genomic integration.

[0095] The nucleic acid construct can be modified to include one or more reporter genes by any suitable method, such as by generating GFP fusions, which can assist in the localization or identification of successful transformation.

[0096] The modified plant or plant cell is capable of producing halogenated hydrocarbons such as bromoform.

[0097] Preparation and use of animal feed

[0098] The resulting modified plant or plant cell can be amplified and propagated in any suitable manner, such as by means of a flow channel, cutting, and seed preparation. The modified plant, whether or not it has undergone further processing, can be used as animal feed or a feed supplement and / or grown at a location where it can be consumed by the animal during normal feeding. As mentioned, the expression of the provided polypeptide can be induced by a suitable method. In the case of production and at the time of production, bromoform or other methane-reducing substances can inhibit methane production in the recipient animal, for example, by inhibiting the microbial production of methane.

[0099] Generally, the animal is a ruminant and can be a bovine, sheep, horse, camel, or deer. The animal can be a dairy cow, sheep, or goat. Given the non-production of energy-intensive methane, the feeding efficiency can be increased. The modified plant or the derived feed can be fed to the animal intermittently or continuously as appropriate.

[0100] Depending on the species and cultivar, the modified plant can be grown as an annual or perennial crop and as a mixture with other suitable species or as a single crop. Its tissue can be used as agricultural food for the livestock to forage on their own. Alternatively, the modified plant tissue can be harvested and processed in any manner suitable for providing feed (or animal feed), including but not limited to hay, straw, silage, grains, legumes, oilseeds, tubers, mixed diets, compressed diets, and pelleted diets. Extracts from the modified plant can also be fed to the livestock in their drinking water or lick blocks, etc. Any means can be used to enable the modified plant material or the extract from the material to enter the rumen of the livestock. The content of bromoform, related halogenated compounds, or other components can be measured before feeding the modified plant material or the extract from the material to the animal to determine the appropriate feeding amount.

[0101] Examples

[0102] The present invention is further illustrated by the following non-limiting examples. The following experiments demonstrate some embodiments of the present invention in practice.

[0103] Example 1 - Transient expression of AtMbb1, CcMbb3, and AaMbb4-2 in maize protoplasts

[0104] Express the Mbb1 protein of Asparagopsis taxiformis (AtMbb1, SEQ ID NO:41), the Mbb3 protein of Chondrus crispus (CcMbb3 SEQ ID NO:6), and the Mbb4-2 protein of Asparagus acutifolius (AaMbb4-2 SEQ ID NO:19) in the maize protoplast system. Design four green fluorescent protein (GFP) fusion constructs for each gene and add signal peptides to direct the expressed fusion proteins to the cytosol, chloroplast, mitochondrion, and peroxisome, respectively. The gene sequences of AtMbb1 (SEQ ID NO:80), AaMbb4-2 (SEQ ID NO:58), and CcMbb3 (SEQ ID NO:52) were codon-optimized for expression in the maize system. Thus, a total of 12 vectors were designed and prepared for transient expression in maize protoplasts as indicated below.

[0105] Construct design: The chloroplast transit peptide (SEQ ID NO:88), mitochondrial targeting peptide (SEQ ID NO:89), and peroxisome targeting peptide (SEQ ID NO:90) were identified from typical maize chloroplast-localized proteins (ribulose bisphosphate carboxylase small subunit 2, GenBank accession: NP_001338725), mitochondrial-localized proteins (superoxide dismutase 3, GenBank accession: NP_001105742), and peroxisome-localized proteins (no ear 1, GenBank accession: NP_001131410). These signal peptides were fused to the N-terminus (chloroplast transit peptide, mitochondrial targeting peptide) or C-terminus (peroxisome targeting peptide) of AtMbb1, AaMbb4-2, and CcMbb3, while GFP (eGFP, SEQ ID NO:97) was fused to the C-terminus of these genes.

[0106] Two linkers (Linker 1 (SEQ ID NO:93) and 3XGGGGS (SEQ ID NO:94)) were used between the domains of the fusion proteins. Signal peptides were not found in AtMbb1, AaMbb4-2, and CcMbb3, indicating that they are cytosolic-localized proteins. Therefore, AtMbb1-GFP, AaMbb4-2-GFP, and CcMbb3-GFP without additional signal peptides were considered to be cytosolic-localized. The chimeric genes were driven by the maize Ubi-1 promoter (SEQ ID NO:91). The gene cassette was cloned into the pUC57 vector. Examples indicating how to construct such constructs can be seen in Figures 1A to 1D Figure 1A ​Shows a cytosolic localization protein, with the gene sequence fused to a 3XG4S linker and GFP, and driven by the maize ubi-1 promoter, and subsequently driven by the NOS terminator (SEQ ID NO:96). The synthetic fragment was cloned into pUC57. Figure 1B Shows a chloroplast localization protein, where the signal peptide from the maize RbcS protein is fused to the N-terminus of the gene of interest, and subsequently fused to GFP. The start codon (ATG) of the gene of interest was removed, and two linkers were added to improve protein expression. The chimeric gene was inserted between the maize ubi-1 promoter and the NOS terminator. Figure 1C Shows a mitochondrial localization protein, where the mitochondrial targeting peptide from maize SOD3 is fused to the N-terminus of the gene of interest, and subsequently fused to GFP. The start codon (ATG) of the gene of interest was removed, and two linkers were added to improve protein expression. The chimeric gene was inserted between the maize ubi-1 promoter and the NOS terminator. Figure 1D Shows a peroxisome localization protein, where the gene of interest is fused to GFP and subsequently fused to a peroxisome targeting peptide. Two linkers were added to improve protein expression. The chimeric gene was inserted between the maize ubi-1 promoter and the NOS terminator. In the context of the present invention, alternative reporter genes, genes (such as those described herein), promoters, terminators, and linkers can of course be used as appropriate, and some components (such as reporter genes) can be completely omitted if needed.

[0107] Protoplast isolation and transfection: Protoplasts were isolated from the leaf tissue of 7-day-old etiolated maize seedlings (Coy et al., 2022. “Protoplast isolation and transfection in maize.” Methods Mol Biol.). Briefly, the first true leaves were collected and the tips and bottoms were removed. The leaf tissue was cut into 0.5 - 1 mm slices using a new lancet and distributed in a petri dish containing the digestion solution. The plate was placed in a vacuum chamber under house vacuum for 30 minutes and then moved to an orbital shaking incubator at 28°C. The leaf tissue was incubated for 4 hours until protoplasts were released. The protoplast solution was collected using a 0.45 μm Steriflip filter device (Millipore) and washed three times by adding 10 mL of MMg solution. The protoplasts were counted using a hemocytometer.

[0108] Received plasmid DNA and transformed it into Escherichia coli. The DNA was extracted and confirmed by PCR. To obtain large amounts of DNA required for protoplast transfection of DNA, a maxiprep was performed. For transfection, 40% polyethylene glycol (PEG) was added to a mixture of MMg solution, plasmid DNA, and protoplasts. Each plasmid was co-transfected with a reference plasmid expressing the fluorescent protein mCherry under the control of a constitutive promoter as a transfection efficiency control. mCherry was used as a positive control for subcellular localization. mCherry is a derivative of RFP and is used for co-localization of GFP fusion proteins. For each target, there is an mCherry control associated with it.

[0109] Finally, the protoplasts were incubated overnight at 28 °C in the dark. The cells were used for localization studies by fluorescence microscopy. Expression of the mCherry gene was used to determine transfection efficiency. Transfection efficiency can be calculated as the percentage of fluorescent protoplasts monitored under a fluorescence microscope out of the total protoplasts. Successful transfection and expression of the fusion construct were confirmed by detecting GFP by fluorescence microscopy.

[0110] Fluorescence microscopy: To determine the expression of the AtMbb1 and CcMbb3 constructs, transient expression analysis was performed by fluorescence microscopy analysis in transfected maize protoplast cells. Maize protoplasts were transfected as described above and then analyzed to determine the subcellular localization of the expressed proteins.

[0111] Imaging was performed 48 hours after transfection and collected by stereomicroscopy to determine appropriate subcellular localization. Images were taken at 20x magnification in the GFP and mCherry channels under the same acquisition settings and then merged to generate Figures 3 and 4. The results of expression and localization by fluorescence microscopy are summarized in Table 1.

[0112] Figure 3 depicts the resulting fluorescence microscopy images of the AtMbb1 construct. Figure 3A Cells transfected with a cytosolic targeting construct are shown; Figure 3B Cells transfected with a chloroplast targeting construct are shown; Figure 3C Cells transfected with a mitochondrial targeting construct are shown; Figure 3D Cells transfected with a peroxisomal targeting construct are shown.

[0113] Figure 4 depicts the resulting fluorescence microscopy images of the CcMbb3 construct. 4A shows cells transfected with a cytosolic targeting construct; 4B shows cells transfected with a chloroplast targeting construct; 4C shows cells transfected with a mitochondrial targeting construct; 4D shows cells transfected with a peroxisomal targeting construct.

[0114] Table 1: Fluorescence microscopy results

[0115]

[0116] Protein expression was confirmed in all AtMbb1 and CcMbb3 transfections. The plasmid sequences of all 12 constructs were confirmed by PCR and digestion. The targeting sequences worked for most constructs.

[0117] Example 2 - Expression of Myriactula bracteata Mbb1 (AtMbb1) in Arabidopsis thaliana

[0118] Four vectors suitable for stable transformation of Myriactula bracteata Mbb1 (AtMbb1, SEQ ID NO:80) were designed and introduced into Arabidopsis thaliana. The gene sequence was codon-optimized for expression in Arabidopsis thaliana. A 3XHA epitope tag (SEQ ID NO:95) was added to the gene to facilitate detection and quantification of the expressed protein. A signal peptide was added to direct Mbb1 expression to chloroplasts, mitochondria, and peroxisomes, respectively. Mbb1 expression was driven by the CaMV 35S promoter (SEQ ID NO:92). As described herein, transgenic plants were further analyzed by molecular analysis to determine whether the plants had been successfully modified, whether the constructs had been successfully transcribed into mRNA, and whether the constructs had been successfully translated into mature protein.

[0119] Construct design: To be consistent with the transient expression experiments of the three proteins described in Example 1, the same signal peptides used in transient expression were used to prepare transgenic Arabidopsis thaliana plants expressing fusion proteins targeted to different organelles. These signal peptides were fused to the N-terminus or C-terminus of AtMbb1, while the 3XHA tag was fused to the C-terminus of this gene. Two linkers (Linker 1 (SEQ ID NO:93) and 3XGGGGS (SEQ ID NO:94)) were used to connect the different components (signal peptide, AtMbb1 protein, and 3XHA tag) to prepare the fusion protein. No signal peptide was found in AtMbb1, indicating that it is a cytosolic localization protein. Therefore, AtMbb1-HA without an additional signal peptide was considered to be cytosolic localization. The coding sequence was codon-optimized for expression in Arabidopsis thaliana, and the synthetic gene cassette was cloned into the pCAMBIA1300-35S vector, where the gene cassette was driven by the enhanced cauliflower mosaic virus 35S promoter.

[0120] In Figures 2A to 2D examples indicating how to construct such constructs can be seen. In the context of the present invention, alternative epitope tags, genes (such as the genes described herein), promoters, terminators, and linkers can of course be used as appropriate, and some components (such as epitope tags) can be completely omitted if necessary. Figure 2AChloroplast-localized AtMbb1 is shown. The signal peptide from the maize RbcS protein was placed at the N-terminus of AtMbb1, followed by 3XHA. The ATG of AtMbb1 was removed and two linkers were added to optimize protein expression. The synthetic cassette was cloned into pCAMBIA1300-35S, where the cassette is driven by the enhanced 35S promoter.

[0121] Figure 2B Mitochondria-localized AtMbb1 is shown. The mitochondrial targeting peptide from maize SOD3 was fused into the N-terminus of AtMbb1, followed by fusion into 3XHA. The ATG of AtMbb1 was removed and two linkers were added to improve protein expression. The synthetic cassette was cloned into pCAMBIA1300-35S, where the cassette is driven by the enhanced 35S promoter.

[0122] Figure 2C Peroxisome-localized AtMbb1 is shown. AtMbb1 was fused with 3XHA and then fused with the peroxisome targeting peptide. Two linkers were added to improve protein expression. The synthetic cassette was cloned into pCAMBIA1300-35S, where the cassette is driven by the enhanced 35S promoter.

[0123] Figure 2D Cytosol-localized AtMbb1 is shown. No signal peptide was found in AtMbb1, indicating that AtMbb1 is a cytosol-localized protein. Therefore, AtMbb1 was fused with 3XG4S linker and 3XHA. The synthetic cassette was cloned into pCAMBIA1300-35S, where the cassette is driven by the enhanced 35S promoter.

[0124] The constructs were introduced into Agrobacterium: The plasmid DNA was transformed into Escherichia coli, the DNA was extracted and confirmed by PCR, and then transformed into Agrobacterium. The completed constructs were introduced into Agrobacterium strain GV3101 by electroporation. The vector identity was confirmed by PCR after transformation.

[0125] Agrobacterium-mediated transformation in Arabidopsis thaliana: Arabidopsis thaliana ecotype Columbia (Col-0) was used for Agrobacterium-mediated transformation by the floral dip method (Clough and Bent, 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J.). Seeds were collected from mature plants after transformation. Seeds were collected from the plants, sterilized and plated on hygromycin selective growth medium. Plants showing elongated hypocotyls after a long dark treatment were transferred to soil for genotyping. Tissue was collected from the rosette leaves of young plants, and DNA was isolated or the tissue was flash frozen for RNA and protein extraction for downstream assays.

[0126] Plant growth phenotypes: Plants were grown and their morphological and growth phenotypes were evaluated. Figure 5 Transgenic plant lines 2007, 2011, and 2012 (boxed and labeled) and six wild-type Arabidopsis plants (unlabeled) are shown. There were no discernible differences between the phenotypes of the plants, confirming that the transgenic plants exhibited normal phenotypes and grew normally.

[0127] Selection of transgenic Arabidopsis thaliana: Screening of transgenic Arabidopsis thaliana was carried out by placing the seeds on a selection medium (half-strength MS) with hygromycin (50 mg / L). After two weeks, the resistant seedlings were transferred to soil.

[0128] Genotyping: Genotyping of transgenic plants was performed by PCR using primers specific for the selectable marker (hyg - hygromycin resistance gene) used to select transgenic events. A total of 74 plants were positive in the presence of the hyg marker by PCR testing, and the results were visualized on an agarose gel. Further analysis was performed on 32 hyg-positive plant lines. In each event, the copy number of the T-DNA present in the Arabidopsis genome was determined by digital droplet PCR analysis, and the results are listed in Table 2 below. DNA was isolated from previous genotyping assays for copy number analysis. Probes targeting the transgene were compared to a gene with a known copy number to obtain a copy number variation (CNV) value relative to the copy number of the known gene. Plants were grouped based on the target signal of the transfection construct. The results confirmed that the plants were successfully modified with the construct.

[0129] Table 2: Plant copy number results

[0130]

[0131] The T2 (second generation) plants derived from the above lines were further genotyped. PCR was performed on the T2 plant material using the same method as described above. These results confirmed that the transgenes present in the T1 (first generation) plants were inherited by the T2 plants.

[0132] Transcription analysis: Then, the levels of Mbb1 transcripts were measured by qRT-PCR in each event. RNA was extracted from rosette leaves and the cDNA concentration was normalized across all samples. The expression of the gene of interest (AtMbb1) was normalized relative to WT-actin using ΔΔCq analysis. The results are shown in Figure 6A -C. Table 3 presents the Figure 6A results for Plate 1 depicted in Figure 6B ; Table 4 presents the Figure 6C results for Plate 2 depicted in Figure 6A ; Table 5 presents the

[0133] results for Plate 3 depicted in

[0134] Plant ID Expression Plant ID Expression 2007 1.6813888 2014 0.0806747 2008 0.0093421 2015 0.0665885 2010 0.1086749 2016 0.1098342 2011 0.8176041 2018 1.4071898 2012 0.3359215 2019 0.0210561 2013 0.020181 2022 1.4357404

[0135] Table 3: Transcription Results for Plate 1

[0136] Plant ID Expression Plant ID Expression 2023 0.17853 2345 0.02670 2024 0.22615 2346 0.00739 2025 0.03234 2347 0.10461 2026 0.83602 2348 0.03683 2027 0.01342 2349 0.18252 2344 0.19992 2350 1.47380

[0137] Table 4: Transcription Results for Plate 2

[0138] Plant ID Expression Plant ID Expression 2030 0.16285 2034 0.41210 2031 0.10667 2035 0.05307 2032 1.00000 2036 0.09461 2033 0.06003 2037 0.41223

[0139] Translation analysis (T1): The production of Mbb1 protein was analyzed by Western blot using an antibody against the HA tag. Transgenic Arabidopsis previously identified as positive for the selection marker was grown in a growth chamber under long-day conditions to increase leaf material for sampling, where seeds were collected and stored. Leaf tissue was quickly frozen in liquid nitrogen and stored at -80°C until needed for total protein extraction. The frozen tissue was homogenized using a bead mill, and proteins were extracted in an extraction buffer and centrifuged to remove excess plant material. Protein concentration was measured by Bradford assay against a known BSA standard. Samples for Western blot analysis were normalized relative to 25 μg total protein, run on a Tris-glycine gel, and transferred to PVDF. Equal loading was determined by Ponceau staining. An antibody raised against the HA-tag was used to detect the protein, where a positive control confirmed correct detection.

[0140] The presence of the transgenically encoded protein was confirmed by Western blotting using the procedures described above, thus confirming successful translation. The lines that tested positive for the protein were: (cytosol-targeted) 2007, 2011, 2012, 2331, 2332, 2333, 2335; (chloroplast-targeted) 2018, 2023, 2026; (mitochondria-targeted) 2350, 3652, 3657, 3660, 3664; (peroxisome-targeted) 2030, 2032, 2038, 2353.

[0141] Translation analysis (T2): Protein expression in T2 (second generation) plants derived from the selected plant lines tested above was further analyzed by Western blotting using the same procedures as those described above. Lines 2007, 2023, 2335, 2350, 2353, and 3652 were submitted for analysis. Subsequently, pools of previously grown 10-day-old T2 seedlings were frozen and stored at -80 °C. Total protein was extracted under native conditions and Western blotting was performed to visualize the transgenically encoded protein in the T2 generation. 75 μg of total extract was loaded for all T2 samples.

[0142] The presence of the transgenically encoded protein was confirmed in all tested lines except 2335, which was indeterminate. These results demonstrate that the transgene can be inherited across generations and can be successfully translated in T2 generation transgenic plants.

[0143] Thus, successful transformation of Arabidopsis plants with the vectors as described has been achieved. It was further shown that the transgenic Arabidopsis plants express the transgene of interest and the mature protein encoded by the transgene has been detected by Western blotting. Thus, it is expected that plants expressing VHPO encoded by the transgene will produce bromoform when grown.

[0144] Example 3 - Liquid culture of Arabidopsis seedlings

[0145] Although it is expected that the transgenic Arabidopsis plants of the present invention (such as the transgenic Arabidopsis plants described in Example 2) can produce bromoform without the addition of any additional substrates or cofactors, the plants can be grown in liquid culture in the presence of substrates and cofactors (such as KBr, sodium orthovanadate, pentane-2,4-dione, and H2O2) to further stimulate and / or increase bromoform production.

[0146] For each transgenic plant sample, prepare approximately 100 seeds in a 1.5 mL Eppendorf tube, add 1 mL of sterile water and mix well. Stand the tube upright at room temperature (23 °C - 25 °C) for 10 minutes. Remove the water and add 1 mL of fresh sterilization solution (5% (v / v) bleach, 0.01% (v / v) sterile water containing Tween 20). Mix gently on an oscillator for 5 - 10 minutes. Discard the sterilization solution and wash with 1 mL of sterile water for 5 minutes. Repeat this step 3 times. This step should be carried out in a laminar flow hood to avoid contamination.

[0147] Keep the surface-sterilized seeds in a sealed tube with 1 mL of sterile water at 4 °C in the dark for 2 - 4 days to promote uniform germination, and then use the seeds in the experiment. Prepare a half-strength Murashige and Skoog (1 / 2 MS) liquid growth medium (Sigma-Aldrich) containing 0.5% w / v glucose (or sucrose) and 0.1% w / v MES buffer. Adjust the pH to 5.7 using 5 M KOH. Autoclave. For each sample, transfer 100 sterile seeds to a sterile 250 mL conical flask containing 50 mL of sterile 1 / 2 MS medium. Cover the flask and incubate the flask on an orbital shaker at approximately 150 rpm in a growth chamber set to standard Arabidopsis growth conditions (20 - 22 °C, 16 hours light [100 μmol m-2 s-1 PPFD], 8 hours dark).

[0148] After 5 - 7 days, a mass of young seedlings should have grown in the flask. Pour out the 1 / 2 MS medium, and then replace it with fresh 1 / 2 MS medium containing the appropriate substrates and cofactors for bromoform production (50 mM KBr, 10 μM sodium orthovanadate, 1 mM pentane-2,4-dione, and 2 mM H2O2), and incubate for up to 5 to 7 days. Then the seedlings can be rinsed with sterile water, blotted dry on paper towels, weighed, frozen in liquid nitrogen, and stored at -80 °C for analysis. Note that the number of seeds / sample and the volume of medium incubation can be scaled down for culturing in smaller containers such as multi-well plates, depending on how much tissue needs to be extracted later.

[0149] Example 4 - Soil Cultures of Arabidopsis thaliana for Leaf Spraying

[0150] Although the transgenic Arabidopsis plants of the present invention (such as the transgenic Arabidopsis plants described in Example 2) are expected to produce bromoform without the addition of any additional substrates or cofactors, the plants can be grown in soil culture and additionally provided with substrates and cofactors (such as KBr, sodium orthovanadate, pentane-2,4-dione, and H2O2) to further stimulate and / or increase bromoform production.

[0151] Grow transgenic Arabidopsis plants approximately 4 - 6 weeks old in soil for 3 weeks. Provide substrates and cofactors by directly spraying a solution of 50 mM KBr, 10 μM sodium orthovanadate, 1 mM pentane-2,4-dione, and 2 mM H2O2 onto the rosette leaves of the plants. Spray the leaves with the solution three times a day for three days. A small amount of surfactant (such as Tween 20) can be added to the solution to aid uptake as the leaf surface is hydrophobic.

[0152] Example 5 - Transgenic Arabidopsis lines produce bromoform in vivo

[0153] Tested in vivo bromoform production in seedlings or rosette leaf tissue from T2 Arabidopsis lines expressing AtMbb1. Bromoform was detected by GC-MS as described by Thapa et al., (2020). Leaf tissue samples collected from transgenic plants were extracted with MeOH overnight. After incubation, the samples were vigorously agitated on a vortex mixer, centrifuged at 16,000×g for 30 minutes to remove debris, and then aliquots of the supernatant were analyzed by GC-MS (1260G with 7890a MS; Agilent Technologies) in electron ionization (70 eV) mode using a DF-5ms ultra-inert GC column (length is 30 m, width is 0.25 mm, and film thickness is 0.5 μM). Frozen tissue samples were extracted at 300 ul / 100 mg tissue, the extracts were centrifuged, and then 50 ul was used for GC / MS analysis. Bromoform production was quantified based on a calibration curve generated from bromoform standards. Column temperature conditions were as follows: 40 °C for 3 minutes, increased to 200 °C at 10 °C / minute, and held for 1 minute, with a total run time of 20 minutes. The injection port, interface, and ion source were maintained at 250 °C, 300 °C, and 230 °C, respectively. Helium was used as the carrier gas at a flow rate of 0.9 mL / minute.

[0154] Bromoform standards were tested against a known concentration of naphthalene (0.5 μg / mL, retention time is 10.263 minutes) and detected between 0.25 μg / mL and 5 μg / mL, where the retention time is 7.905 minutes. 0.5 μg / mL naphthalene was included as an internal standard in all test samples. Each sample was extracted in chloroform spiked with 0.5 μg / mL naphthalene, centrifuged, and 50 μL of the supernatant was passed through the GCMS.

[0155] T2 Leaf Samples: T2 plants of each of the 19 different Arabidopsis events expressing AtMbb1 were grown in soil for three weeks. Leaves from these T2 plants grown according to Example 4 were harvested, frozen, and analyzed for the presence of bromoform by gas chromatography - mass spectrometry (GCMS). The samples were subjected to a double extraction and concentration process. The samples were initially extracted with chloroform as described above. The samples were then re - extracted with 150 μL of saturated brine and 50 μL of methanol, vortexed to separate the phases, and the upper chloroform and methanol phases were transferred to a clean tube. The lower phase was re - extracted with 300 μL of methyl tert - butyl ether, and the top layer was transferred to the chloroform / methanol. The samples were then dried under nitrogen and immediately redissolved in 50 μL of methanol for GCMS injection. Bromoform was detected in a total of 4 events (2007, 2011, 2012, and 2023), while no bromoform was detected in wild - type Arabidopsis plants. Peaks were detected at retention times of 7.922 (2007), 7.913 (2011), and 7.918 (2012 and 2023) minutes, which is consistent with the elution of bromoform. Some samples were reinjected in a different manner to confirm that the positive results were not due to reagent contamination or carry - over. Lines 2007, 2011, and 2012 express cytoplasmic AtMbb1 (without a signal sequence), while AtMbb1 is targeted to the chloroplast in event 2023.

[0156] Figure 7A -D is the gas chromatogram of the T2 leaf samples; for each sample, two chromatograms are presented with x - axes of different scales to better visualize the data. The sample material was spiked with an internal standard, and thus peaks for bromoform and naphthalene at the elution times are visible in each sample chromatogram. The peaks labeled with the elution times are related to the sample material of the combined extracted ion chromatogram. Figure 7E is the chromatogram of the positive control: 0.25 μg / mL bromoform standard. Figure 7A is the chromatogram of the 2007 sample. Figure 7B is the chromatogram of the 2011 sample. Figure 7C is the chromatogram of the 2012 sample. Figure 7D is the gas chromatogram of the 2023 sample.

[0157] The mass spectra of the samples were then analyzed to confirm the identity of the bromoform peaks. As an example, the mass spectrum from event 2007 along with the NIST reference spectrum of bromoform is shown in Figure 8 The 2007 sample mass spectrum is as shown above, and the NIST reference spectrum is as shown below. The 2007 sample spectrum shows a strong match with the expected spectrum of bromoform, where the complete and partial bromoform ions have corresponding peaks. This confirms the presence of bromoform in the 2007 sample, initially detected by gas chromatography.

[0158] T2 Seedling Samples: Bromoform was also detected in the plant material taken from T2 seedlings grown according to Example 3 and was tested according to the same protocol as described in Example 5 above. In addition to the transgenic T2 seedlings, wild-type (non-transgenic) Arabidopsis seedlings were analyzed in parallel as a negative control. An extract of Asparagopsis taxiformis was used as a positive control. Commercial bromoform was used as a standard. A bromoform peak was observed by gas chromatography, and a peak matching the ion of full bromoform was detected by mass spectrometry in the T2 seedling samples.

[0159] In summary, in these examples, it has been shown that algal VHPO enzymes can be expressed in functional form in plant cells, and transgenic plants expressing such VHPO enzymes are capable of producing bromoform in vivo.

[0160] Table 6: List of Identified Sequences

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214] Examples of the inventive concept are stated in the following clauses.

[0215] 1. A nucleic acid construct for inducing the expression of a polypeptide in a plant cell, the nucleic acid construct comprising a coding sequence encoding a polypeptide comprising a vanadate-dependent haloperoxidase (VHPO) or a functional fragment or homolog thereof;

[0216] wherein the coding sequence is operably linked to one or more regulatory elements adapted to drive the expression of the polypeptide in a plant cell.

[0217] 2. A modified plant cell comprising a nucleic acid coding sequence encoding a polypeptide comprising a vanadate-dependent haloperoxidase (VHPO) or a functional fragment or homolog thereof.

[0218] 3. The modified plant cell according to clause 2, wherein the modified plant cell comprises the construct according to clause 1.

[0219] 4. A plant comprising the modified plant cell according to clause 2.

[0220] 5. The plant according to clause 4, wherein the plant expresses VHPO in an edible part of livestock (appropriately in its leaves or grains).

[0221] 6. The plant or plant cell according to any one of clauses 2 to 5, wherein the plant or the plant cell produces brominated hydrocarbons, appropriately bromoform.

[0222] 7. The nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the VHPO polypeptide is derived from a species selected from the following: Asparagopsis (e.g., Asparagopsis taxiformis, Asparagopsis armata), deep-sea unicellular cyanobacteria, Ecklonia, Ascophyllum nodosum, Alteromonas naphthalenivorans; Caulerpa (e.g., Caulerpa taxifolia); Chaetomorpha (e.g., Chaetomorpha linum), Chondrus crispus, Cystoseira, Corallina officinalis, Jania rubens, Phaeophyceae, Chondrus crispus, Gracilaria (e.g., Gracilaria changii, Gracilaria vermiculophylla); Cystoseira trinodis, Hypnea pannosa, Laminaria (e.g., Laminaria digitata, Saccharina japonica), Laurencia filamentosa, Macrocystis pyrifera, Sargassum, Padina pavonica, Cladophora braunii, Dictyota dichotoma, Gigartina stellata, Oedogonium, Padina australis, Ulva lactuca and Ulva (e.g., Enteromorpha intestinalis, Enteromorpha linza, Ulva lactuca).

[0223] 8. The nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the VHPO polypeptide is derived from a microalgal species or a macroalgal species.

[0224] 9. The nucleic acid construct, plant cell or plant according to clause 8, wherein the microalgal species is a member of the genus Emiliania, Calcidiscus or Chaetoceros.

[0225] 10. The nucleic acid construct, plant cell or plant according to clause 8, wherein the macroalgal species is a member of the genus Asparagopsis or Chondrus.

[0226] 11. The nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the VHPO polypeptide is derived from Asparagopsis armata or Asparagopsis taxiformis.

[0227] 12. The nucleic acid construct, plant cell or plant according to clause 11, wherein the VHPO polypeptide is derived from Asparagopsis taxiformis from the Azores, Asparagopsis armata from the Azores or Asparagopsis armata from Ireland.

[0228] 13. The nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the VHPO is vanadate-dependent bromoperoxidase (VBPO).

[0229] 14. The nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the VBPO is selected from Mbb1, Mbb3 and Mbb4 of Asparagopsis taxiformis.

[0230] 15. The nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the nucleic acid is DNA.

[0231] 16. The nucleic acid construct, plant cell or plant according to clause 15, wherein the DNA is in the form of a plasmid.

[0232] 17. A nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the coding sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NOs: 47, 49 to 54, 57 to 60, 64 to 67, 71 to 74, 77 to 80 and 82 to 87.

[0233] 17a. A nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the construct comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NOs: 52, 58 or 80.

[0234] 18. A nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the coding sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NOs: 47, 49 and 50 (Mbb1, Mbb3 or Mbb4 of Asparagopsis taxiformis).

[0235] 19. A nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the coding sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NOs: 54, 57 to 60, 64 to 67, 71 to 74 and 77 to 79 (VHPO of Asparagopsis armata).

[0236] 20. A nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the polypeptide comprising the VHPO comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NOs: 1, 3 to 15, 18 to 21, 25 to 28, 32 to 35, 38 to 41 and 43 to 46.

[0237] 21. A nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the polypeptide comprising the VHPO comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of Mbb1, Mbb3 and Mbb4 of Asparagopsis taxiformis (SEQ ID NOs: 1, 3 and 4).

[0238] 22. A nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the polypeptide comprising the VHPO comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NOs: 15, 18 to 21, 25 to 28, 32 to 35 and 38 to 40 (VHPO of Asparagopsis armata).

[0239] 23. A nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the polypeptide comprising the VHPO comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of the vBPOs of Chondrus crispus (SEQ ID NOs: 5 to 7).

[0240] 24. A nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the polypeptide comprising the VHPO comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of the following: vBPO of deep - sea unicellular cyanobacteria (SEQ ID NO: 8); vBPO of Alteromonas naphthalenivorans (SEQ ID NO: 9), vBPO of Ascophyllum nodosum (SEQ ID NO: 10), vBPO of Corallina officinalis (SEQ ID NO: 11), Corallina pilulifera (SEQ ID NO: 12), vBPO of Gracilaria changii (SEQ ID NO: 13) and vBPO of Laminaria digitata (SEQ ID NO: 14).

[0241] 25. A nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the plant or the plant cell is a terrestrial plant.

[0242] 26a. A nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the plant or the plant cell is a monocotyledonous plant.

[0243] 26b. A nucleic acid construct, plant cell or plant according to clause 26a, wherein the monocotyledonous plant is selected from maize, millet, sorghum or forage grass.

[0244] 27. A nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the plant or the plant cell is a dicotyledonous plant.

[0245] 28. A nucleic acid construct, plant cell or plant according to clause 27, wherein the dicotyledonous plant is alfalfa or clover.

[0246] 29. A nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the coding sequence is operably linked to a promoter to drive the expression of the polypeptide in the plant cell.

[0247] 30. A nucleic acid construct, plant cell or plant according to clause 29, wherein the promoter is a constitutive promoter or an inducible promoter.

[0248] 31. A nucleic acid construct, plant cell or plant according to clause 29 or 30, wherein the promoter is selected from AlcR / AlcA (ethanol inducible); GR fusion, GVG and pOp / LhGR (dexamethasone inducible); XVE / OlexA (β-estradiol inducible); and heat shock inducible.

[0249] 32. A nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the VHPO is monomeric.

[0250] 33. A nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the coding sequence is operably linked to a nucleic acid sequence encoding a targeting signal.

[0251] 34. A nucleic acid construct, plant cell or plant according to any one of the preceding clauses, wherein the polypeptide comprises a targeting signal.

[0252] 35. A nucleic acid construct, plant cell or plant according to clause 33 or 34, wherein the targeting signal is a peroxisome targeting signal, optionally wherein the peroxisome targeting signal is SEQ ID NO:90.

[0253] 36. A nucleic acid construct, plant cell or plant according to clause 33 or 34, wherein the targeting signal is a mitochondrial targeting signal, optionally wherein the mitochondrial targeting signal is SEQ ID NO:89.

[0254] 37. A nucleic acid construct, plant cell or plant according to clause 33 or 34, wherein the targeting signal is a chloroplast targeting signal, optionally wherein the chloroplast targeting signal is SEQ ID NO:88.

[0255] 38. An animal feed or animal supplement comprising a modified plant cell or plant according to any one of clauses 2 to 37.

[0256] 39. A method for reducing methane production in an animal, the method comprising growing or culturing a modified plant or plant cell according to any one of clauses 2 to 37 under conditions suitable for producing halogenated hydrocarbons, and feeding an effective amount of the modified plant or plant cell to the animal.

[0257] 39b. The method according to clause 39, wherein the plant is subjected to stress (such as heat shock or drought).

[0258] 39c. The method according to clause 39, wherein the growth or the cultivation of the modified plant or plant cell does not include providing a substrate or a cofactor related to the production of a halogenated hydrocarbon.

[0259] 39d. The method according to clause 39, wherein the growth or the cultivation of the modified plant or plant cell includes providing a substrate or a cofactor related to the production of a halogenated hydrocarbon.

[0260] 39e. The method according to clause 39c or clause 39d, wherein the halogenated hydrocarbon is a brominated hydrocarbon.

[0261] 39f. The method according to clause 39e, wherein the brominated hydrocarbon is bromoform.

[0262] 39g. The method according to any one of clauses 39c, 39d, 39e or 39f, wherein the substrate or the cofactor related to the production of the halogenated hydrocarbon includes at least one compound selected from the group consisting of: KBr, sodium orthovanadate, pentane-2,4-dione and H2O2.

[0263] 40. A method for reducing methane production in an animal, the method comprising administering to the animal an effective amount of a modified plant cell, a plant or an animal feed according to any one of clauses 2 to 37.

[0264] 41. The method according to clause 39 or 40, wherein the animal is a ruminant.

[0265] 42. The method according to any one of clauses 39 to 41, wherein the animal is a bovine.

[0266] 43. The method according to any one of clauses 39 to 42, wherein the animal is a dairy cow, a sheep or a goat.

[0267] 44. The method according to any one of clauses 39 to 43, wherein methane production is reduced.

[0268] 45. The method according to any one of clauses 39 to 44, wherein the feeding efficiency is improved.

[0269] 46. A method for preparing a modified plant cell, the method comprising:

[0270] transforming a plant cell with a nucleic acid construct according to any one of clauses 1 to 37.

[0271] 46b. A method for preparing a modified plant cell, the method comprising:

[0272] Transfecting a plant cell with a plasmid comprising a nucleic acid construct according to any one of clauses 1 to 37.

[0273] 47. The method according to clause 46 or 46b, wherein the plant cell is transformed by Agrobacterium-mediated infiltration.

[0274] 48. The method according to clause 47, wherein the plant cell is transformed by particle bombardment.

[0275] 48b. The method according to clause 46, wherein the plant cell is transformed by using: CRISPR / Cas system, zinc finger nuclease, TALEN, lentivirus, adenovirus, AAV and / or meganuclease.

[0276] 49. A method for producing an animal feed for reducing methane production in an animal, the method comprising:

[0277] Growing the modified plant cell or plant according to any one of clauses 2 to 37; and

[0278] Processing the modified plant cell or plant into an animal feed.

Claims

1. A nucleic acid construct for inducing the expression of a polypeptide in a plant cell, said nucleic acid construct comprising a coding sequence encoding a polypeptide comprising a vanadate-dependent haloperoxidase (VHPO) or a functional fragment or homolog thereof; wherein said coding sequence is operably linked to one or more regulatory elements adapted to drive the expression of said polypeptide in a plant cell.

2. A modified plant cell comprising a nucleic acid coding sequence encoding a polypeptide comprising a vanadate-dependent haloperoxidase (VHPO) or a functional fragment or homolog thereof, preferably wherein said modified plant cell comprises the construct according to claim 1.

3. A plant comprising the modified plant cell according to claim 2.

4. The plant or plant cell according to claim 2 or claim 3, wherein said plant or said plant cell produces brominated hydrocarbons, suitably bromoform.

5. The nucleic acid construct, plant cell or plant according to any one of the preceding claims, wherein the VHPO polypeptide is derived from a species selected from the following: Asparagopsis spp. (e.g., A. taxiformis, A. armata), Acaryochloris marina, Alaria esculenta, Ascophyllum nodosum, Alteromonas naphthalenivorans; Caulerpa spp. (e.g., Caulerpa taxifolia); Chaetomorpha spp. (e.g., Chaetomorpha linum), Chondrus crispus, Colpomenia sinuosa, Corallina officinalis, Corallina pilulifera, Cystoseira trinodis, Furcellaria spp., Gracilaria spp. (e.g., Gracilaria changii, Gracilaria vermiculophylla); Hormophysa triquetra, Hypnea pannosa, Laminaria spp. (e.g., L. digitata, L. saccharina), Laurencia filiformis, Macrocystis pyrifera, Sargassum flavicans, Zonaria farlowii, Cladophora patentiramea, Dictyota bartayresii, Gigartina spp. (e.g., Gigartina stellata), Oedogonium spp., Padina spp. (e.g., Padina australis), Pterocladia capillacea, and Ulva spp. (e.g., U.(U. intestinalis), Enteromorpha linza, Ulva lactuca).

6. The nucleic acid construct, plant cell or plant according to any one of the preceding claims, wherein the VHPO polypeptide is derived from a microalgal species or a macroalgal species.

7. The nucleic acid construct, plant cell or plant according to claim 6, wherein said macroalgal species is a member of the genus Asparagopsis or Chondrus.

8. The nucleic acid construct, plant cell or plant according to any one of the preceding claims, wherein said VHPO is a vanadate-dependent bromoperoxidase (VBPO).

9. The nucleic acid construct, plant cell or plant according to any one of the preceding claims, wherein said coding sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NOs: 47, 49 to 54, 57 to 60, 64 to 67, 71 to 74, 77 to 80 and 82 to 87.

10. The nucleic acid construct, plant cell or plant according to any one of the preceding claims, wherein said coding sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NOs: 47, 49 and 50 (Mbb1, Mbb3 or Mbb4 of Asparagopsis taxiformis).

11. The nucleic acid construct, plant cell or plant according to any one of the preceding claims, wherein said coding sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NOs: 54, 57 to 60, 64 to 67, 71 to 74 and 77 to 79 (VHPO of Asparagopsis armata).

12. The nucleic acid construct, plant cell or plant according to any one of the preceding claims, wherein the polypeptide comprising the VHPO comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 1, 3 to 15, 18 to 21, 25 to 28, 32 to 35, 38 to 41 and 43 to 46.

13. The nucleic acid construct, plant cell or plant according to any one of the preceding claims, wherein the polypeptide comprising the VHPO comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NO: 1, 3 and 4 (Mbb1, Mbb3 and Mbb4 of Asparagopsis taxiformis).

14. The nucleic acid construct, plant cell or plant according to any one of the preceding claims, wherein the polypeptide comprising the VHPO comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NO: 15, 18 to 21, 25 to 28, 32 to 35 and 38 to 40 (VHPO of Asparagopsis acanthocarpa).

15. The nucleic acid construct, plant cell or plant according to any one of the preceding claims, wherein the polypeptide comprising the VHPO comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of SEQ ID NO: 5 to 7 (vBPO of Chondrus crispus).

16. The nucleic acid construct, plant cell or plant according to any one of the preceding claims, wherein the polypeptide comprising the VHPO comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to any one of the following: vBPO of deep - sea unicellular cyanobacteria (SEQ ID NO: 8); vBPO of Alteromonas naphthalenivorans (SEQ ID NO: 9), vBPO of Ascophyllum nodosum (SEQ ID NO: 10), vBPO of Corallina officinalis (SEQ ID NO: 11), Corallina pilulifera (SEQ ID NO: 12), vBPO of Gracilaria changii (SEQ ID NO: 13) and vBPO of Laminaria digitata (SEQ ID NO: 14).

17. The nucleic acid construct, plant cell or plant according to any one of the preceding claims, wherein the plant or the plant cell is a terrestrial plant.

18. The nucleic acid construct, plant cell or plant according to any one of the preceding claims, wherein the plant or the plant cell is a monocotyledonous plant, preferably wherein the monocotyledonous plant is selected from maize, millet, sorghum or forage grass.

19. A nucleic acid construct, plant cell or plant according to any one of the preceding claims, wherein the plant or the plant cell is a dicotyledonous plant, preferably wherein the dicotyledonous plant is alfalfa or clover.

20. A nucleic acid construct, plant cell or plant according to any one of the preceding claims, wherein the coding sequence is operably linked to a promoter to drive expression of the polypeptide in a plant cell.

21. A nucleic acid construct, plant cell or plant according to any one of the preceding claims, wherein the coding sequence is operably linked to a nucleic acid sequence encoding a targeting signal, and / or wherein the polypeptide comprises a targeting signal.

22. A nucleic acid construct, plant cell or plant according to claim 21, wherein: i) the targeting signal is a peroxisomal targeting signal, optionally wherein the peroxisomal targeting signal is SEQ ID NO: 90; ii) wherein the targeting signal is a mitochondrial targeting signal, optionally wherein the mitochondrial targeting signal is SEQ ID NO: 89; or iii) wherein the targeting signal is a chloroplast targeting signal, optionally wherein the chloroplast targeting signal is SEQ ID NO:

88.

23. An animal feed or animal supplement comprising a modified plant cell or plant according to any one of claims 2 to 22.

24. A method for reducing methane production in an animal, the method comprising growing or culturing a modified plant or plant cell according to any one of claims 2 to 22 under conditions suitable for producing halogenated hydrocarbons, and feeding an effective amount of the modified plant or plant cell to the animal.

25. A method for reducing methane production in an animal, the method comprising administering an effective amount of a modified plant cell, plant or animal feed according to any one of claims 2 to 23 to the animal.

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