Genetically modified endophyte

Abstract

The present invention relates to the delivery of a methane inhibitor to a plant or a grazing animal. This is achieved by infecting grass with an endophyte which has been modified to produce the methane inhibitor in planta. The endophyte produces the methane inhibitor in planta, which may be consumed by the grazing animal.

Description

GENETICALLY MODIFIED ENDOPHYTETECHNICAL FIELD

[0001] The present application generally relates to a method of delivering a methane inhibitor via a genetically modified endophyte. The engineered endophytes may be useful as a method of delivering an agricultural product, e.g., for production of a methane inhibitor within a grassendophyte symbiosis.BACKGROUND

[0002] Approximately 60% of global methane emissions are a result of human activity with agriculture the single largest contributor. In countries that rely heavily on pastoral agriculture, enteric fermentation in ruminants accounts for nearly all of agricultural methane emissions. Ruminant methane production can be attenuated by supplementation of animal feed with methane inhibitor compounds such as lovastatin. However, this approach presents both economic and feasibility challenges for pasture-based agriculture systems.

[0003] Ruminants obtain their primary energy source from volatile fatty acids (VFAs) which are produced by symbiotic rumen microorganisms via fermentation of plant polysaccharides in a ruminant’s diet (Morgavi et al., 2010). As by-products of this fermentation process, hydrogen and carbon dioxide gases are released. The levels of these by-products, which can be inhibitory to the growth of rumen microorganisms, are kept in check by methanogenic archaea which utilise these compounds in anaerobic respiration to generate energy and methane (Moran et al., 2005).

[0004] Methanogenic archaea are the only organisms known to produce methane (Hook et al., 2010; Schafer et al., 1999) and their growth can be inhibited by statins which act as competitive inhibitors of the enzyme HMG-CoA reductase (HMGR), the rate-controlling enzyme of the mevalonate pathway. Inhibition of this pathway in humans prevents the biosynthesis of isoprenoids such as cholesterol, leading to the primary clinical indication of statins as lipid- lowering agents. In archaea, the mevalonate pathway is essential for the biosynthesis of lipids, including archaeol and caldarcheol that comprise the distinctive archaeal cell membrane (Gottlieb et al., 2016; Jain et al., 2014). In contrast, bacteria have different cell membrane compositions and structures and instead rely largely on the MEP (methylerythritol-4-phosphate) pathway for synthesis of their cell membrane lipids (Perez-Gil & Rodriguez-Concepcion, 2013).This distinction provides a critical avenue for statins to act as selective inhibitors of methanogens with minimal impact on other rumen bacteria. This effect was first demonstrated in vitro with lovastatin and mevastatin, which were found to inhibit the growth of Methanobrevibacter without affecting the growth of other rumen bacteria (Miller & Wolin, 2001). In vivo animal studies have also observed reduction in methanogenesis in ruminants such as sheep and goats by supplementation of lovastatin in feed (Candyrine et al., 2018; Mohd Azlan et al., 2018; Morgavi et al., 2013).

[0005] Lovastatin was first discovered in the soil fungus Aspergillus terreus (Alberts et al., 1980), the biosynthetic pathway begins with the iterative condensation of nine acetate (acetyl- CoA and malonyl-CoA) molecules, performed by the nonaketide synthase LovB and the enoyl reductase LovC (Auclair et al., 2001; Hendrickson et al., 1999; Kennedy et al., 1999; Ma et al., 2009; Ma & Tang, 2007). After ~35 reactions, the release of the dihydromonacolin L (DHML) product from LovB is facilitated by the multifunctional esterase LovG (Xu et al., 2013). The cytochrome P450 monooxygenase LovA then sequentially oxidises DHML to monacolin L and monacolin L to monacolin J (Barriuso et al., 2011). LovF, a diketide synthase, generates an a- methylbutyryl side chain from acetyl-CoA and malonyl-CoA, which is finally attached to monacolin J by the acyl transferase LovD to generate lovastatin (Xie et al., 2009).

[0006] The industrial production of lovastatin is primarily performed using liquid-submerged fermentation or solid-state fermentation of terreus strains (Barrios-Gonzalez et al., 2020). For the purpose of methane reduction and global warming mitigation, however, cost remains a restrictive factor for the larger scale integration of lovastatin into animal feed (Abrego-Gacia et al., 2021).

[0007] Accordingly, it is an object of the present invention to go some way to avoiding the above disadvantages; and / or to at least provide the public with a useful choice.

[0008] Other objects of the invention may become apparent from the following description which is given by way of example only.

[0009] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date.SUMMARY OF THE INVENTION

[0010] In a first aspect, the invention provides a method for preparing a modified endophyte capable of producing a methane inhibitor, wherein the method comprises the step of inserting genetic elements into an endophyte to provide the modified endophyte, wherein the genetic elements comprise gene coding sequences, promoters, terminators, and other regulatory elements for selective biosynthesis of the methane inhibitor in planta within a host plant.

[0011] In a second aspect, the invention relates to use of a modified endophyte for delivering a methane inhibitor to a host plant, comprising infecting the plant, or a precursor thereof, with the modified endophyte, wherein the modified endophyte produces the methane inhibitor in planta.

[0012] In a third aspect, the invention provides a modified endophyte capable of producing a methane inhibitor, wherein the modified endophyte comprises genetic elements comprising gene coding sequences, promoters, terminators, and other regulatory elements for selective biosynthesis of the methane inhibitor in planta within a host plant.

[0013] In a fourth aspect, the invention provides a method of delivering a methane inhibitor to a host plant, the method comprising infecting the host plant, or a precursor thereof, with a modified endophyte capable of producing the methane inhibitor, and wherein the methane inhibitor is produced in planta.

[0014] In a fifth aspect, the invention provides a method for reducing methane emissions from an animal, the method comprising: a. providing a host plant infected with a modified endophyte capable of producing a methane inhibitor, wherein the modified endophyte produces the methane inhibitor in plan la b. feeding the plant comprising the methane inhibitor to the animal.

[0015] In a sixth aspect, the invention provides a method for reducing methane emissions from an animal, the method comprising:a. growing a host plant on a substrate, wherein the host plant is infected with a modified endophyte capable of producing a methane inhibitor and the modified endophyte produces the methane inhibitor in planter, b. allowing the animal to graze on the plant on the substrate.

[0016] In some embodiments, the host plant is provided on a substrate.

[0017] In some embodiments, step (b) comprises grazing the animal on the host plant, e.g., wherein the host plant is on a substrate, e.g. wherein the substrate is pasture land or arable soil.

[0018] In some embodiments, step (b) comprises harvesting the host plant and feeding the harvested plant to the animal.

[0019] In some embodiments, the animal is a livestock animal; optionally, wherein the livestock animal is a cattle, sheep, goat, deer, donkey, horse, bison, yak or mule. In some embodiments, the animal is a ruminant. In some embodiments, the ruminant is a cattle, sheep, goat, deer, bison or yak.

[0020] In some embodiments, the host plant is a grass. In some embodiments, the grass belongs to any one of the genera Lolium, Festuca, Poa, Dactylis, Agrostis and Phleum. In some embodiments, the host plant is a pasture grass. In some embodiments, the host plant is a ryegrass.

[0021] In some embodiments, the host plant is a living plant.

[0022] In some embodiments, the modified endophyte is a modified bacterial endophyte or a modified fungal endophyte. In some embodiments, the modified fungal endophyte belongs to the genus Epichloe; e.g., wherein the modified fungal endophyte is Epichloe festucae.

[0023] In some embodiments, the genetic elements comprise gene coding sequences, promoters, and terminators for selective biosynthesis of the methane inhibitor in planta with a host plant. In some embodiments, the genetic elements comprise gene coding sequences for selective biosynthesis of the methane inhibitor in planta with a host plant.

[0024] In some embodiments, the modified endophyte comprises genetic elements for heterologous expression of biosynthetic genes. In some embodiments, the genetic elements comprise gene coding sequences for biosynthesis of the methane inhibitor. In someembodiments, the genetic elements comprise gene coding sequences amplified from biological material or produced synthetically for biosynthesis of the methane inhibitor. In some embodiments, the genetic elements comprise promoter and terminator sequences selective for in planta expression of the methane inhibitor. In some embodiments, the genetic elements comprise promoter, terminator, and other regulatory sequences, amplified from the host endophyte, other biological sources, or produced synthetically, selective for in planta expression of the methane inhibitor. In some embodiments, the gene sequences for biosynthesis of the methane inhibitor are lov gene sequences. In some embodiments, the heterologous gene sequences for biosynthesis of the methane inhibitor are lovB, lovC, and lovG.

[0025] In some embodiments, the genetic elements comprise gene sequences with heterologous promoters and terminators. In some embodiments, the genetic elements comprise a promoter sequence and a terminator sequence from Aspergillus nidulans. In some embodiments, the genetic elements comprise Ptefl promoter sequence and a TtubB terminator sequence from Aspergillus nidulans. In some embodiments, the genetic elements comprise lov gene coding sequences and heterologous promotors and terminators. In some embodiments, the genetic elements comprise lovA, lovB, lovC, lovD, lovF, lovG or a combination of any two or more thereof and heterologous promotors and terminators. In some embodiments, the genetic elements comprise lovB, lovC, and lovG and heterologous promotors and terminators. In some embodiments, the genetic elements comprise lovA, lovD and lovF and heterologous promotors and terminators.

[0026] In some embodiments, the heterologous gene sequences for biosynthesis of the methane inhibitor are under regulatory control of endogenous promotors and terminators. In some embodiments, the heterologous gene sequences for biosynthesis of the methane inhibitor are under regulatory control of Itm promotors and terminators. In some embodiments, the heterologous gene sequences for biosynthesis of the methane inhibitor are lov gene sequences under regulatory control of Itm promotors and terminators. In some embodiments, the heterologous gene sequences for biosynthesis of the methane inhibitor are selected from lovA, lovB, lovC, lovD, lovF, lovG or a combination of any two or more thereof under regulatory control of Itm promotors and terminators. In some embodiments, the heterologous gene sequences for biosynthesis of the methane inhibitor are lovB, lovC, and lovG under regulatory control of Itm promotors and terminators. In some embodiments, the heterologous gene sequences for biosynthesis of the methane inhibitor are lovA, lovD and lovF under regulatory control of Itm promotors and terminators.

[0027] In some embodiments, the modified endophyte comprises a Itm promotor and a Itm terminator.

[0028] In some embodiments, the modified endophyte selectively expresses the methane inhibitor in planta with the host plant. In some embodiments, the genetic elements are host specific and only active when the endophyte is in symbiosis with the host. In some embodiments, there is nil or negligible expression of the methane inhibitor genes or production of the methane inhibitor when the modified endophyte is not growing endophy ti cal ly within the host plant.

[0029] In some embodiments, the modified endophyte produces the methane inhibitor in planta at a rate of at least 0.005 mg / kg host plant (dry weight). In some embodiments, the modified endophyte produces the methane inhibitor in planta at a rate of at least 0.01 mg / kg host plant (dry weight).

[0030] In some embodiments, the endophyte is modified to reduce toxicity caused by the methane inhibitor.

[0031] In some embodiments, the genetic elements enable extracellular localisation of the methane inhibitor. In some embodiments, localisation of the methane inhibitor by the modified endophyte is substantially extracellular. In some embodiments, there is nil or negligible intracellular accumulation of the methane inhibitor.

[0032] In some embodiments, the genetic elements are inserted into the endophyte as cDNA. In some embodiments, the gene coding sequences are inserted into the endophyte as cDNA.

[0033] In some embodiments, the genetic elements inserted into the endophyte are between 1 kb and 60 kb. In some embodiments, the genetic elements inserted into the endophyte are between 5 kb and 60 kb, 10 kb and 60 kb, 15 kb and 60 kb, 20 kb and 60 kb, 25 kb and 60 kb, 30 kb and 60 kb, 35 kb and 60 kb, 40 kb and 60 kb, 45 kb and 60 kb, 50 kb and 60 kb, or 55 kb and 60 kb. In some embodiments, the genetic elements inserted into the endophyte are at least 1 kb. In some embodiments, the genetic elements inserted into the endophyte are at least 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, 50 kb, 55 kb or 60 kb. In some embodiments, the genetic elements inserted into the endophyte are at least 40 kb, 45 kb, 50 kb, 55 kb or 60 kb.

[0034] In some embodiments, the genetic elements comprise a gene sequence encoding an efflux pump. In some embodiments, the genetic elements comprise a gene sequence that confersresistance to the methane inhibitor. In some embodiments, the genetic elements comprise a gene sequence that encodes a reductase that confers resistance to the methane inhibitor. In some embodiments, the genetic elements comprise a gene sequence that encodes a lovastatin-resistant HMG-CoA reductase.

[0035] In some embodiments, the genetic elements comprise:• a gene sequence for biosynthesis of the methane inhibitor, under regulatory control of endogenous promotors and terminators, such as Itm promotors and terminators in E. feslucae• a gene sequence encoding an efflux pump;• a gene sequence that confers resistance to the methane inhibitor, such as a heterologous gene sequence that encodes a reductase that confers resistance to the methane inhibitor.

[0036] In some embodiments, the methane inhibitor is localised extracellularly in the modified endophyte.

[0037] Is some embodiments, the endophyte is modified using MIDAS.

[0038] In some embodiments, the methane inhibitor is a methanogen inhibitor. In some embodiments, the methane inhibitor is any one of a statin, a haemanthamine, a bromoform, a bacteriocin, a non-ribosomal peptide, a flavonoid, a nitro compound, or any methane inhibitor or precursor thereof that can be biosynthesised in a biological cell.

[0039] In some embodiments, the methane inhibitor is lovastatin.

[0040] In some embodiments, the endophyte is an isolated cell.

[0041] In a seventh aspect, the invention provides genetic elements specific for the targeted heterologous in planta expression of methane inhibitor biosynthesis genes in an endophyte for the in planta-specific production of the methane inhibitor.

[0042] In an eighth aspect, the invention provides a plant, or precursor thereof, comprising a modified endophyte according to the third aspect, e.g., wherein the precursor thereof is a seed or seedling.

[0043] In a ninth aspect, the invention provides a seed or seedling infected with a modified endophyte according to the third aspect, e.g., wherein the seed is a ryegrass seed infected with the modified endophyte.

[0044] In a tenth aspect, the invention provides a culture comprising a modified endophyte according to the third aspect.

[0045] In an eleventh aspect, the invention provides an animal feed product comprising a plant product and a methane inhibitor.

[0046] In some embodiments, the plant product is silage, hay, straw or pellets.

[0047] Any of the aforementioned features or embodiments or aspects may be combined with one or more of the other features or embodiments or aspects as described herein.

[0048] This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0049] In addition, where features or aspects of the invention are described in terms of Markush groups, those persons skilled in the art will appreciate that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0050] As used herein “(s)” following a noun means the plural and / or singular forms of the noun.

[0051] As used herein the term “and / or” means “and” or “or” or both.

[0052] The term “comprising” as used in this specification means “consisting at least in part of’. When interpreting each statement in this specification that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner.

[0053] The term “methane inhibitor” as used herein refers to a compound that reduces methane emissions from an animal when ingested by the animal. For example, the methane inhibitor may be a methanogen inhibitor, i.e. a compound that inhibits the activity ofmethanogens. Without wishing to be bound by theory, methanogens are microorganisms that may live in the digestive tract of animals, e.g., ruminants. Methanogens cause the production of methane, i.e. methanogenesis, that is subsequently emitted from the animal. The terms “methane inhibitor” and “methanogen inhibitor” as used herein include compounds that are metabolised by the animal to form the active compound, e.g., a compound that is metabolised to form a methanogen inhibitor.

[0054] The term “livestock animal” as used herein includes, but is not limited to, ruminant species such as a cattle, sheep, goat, deer, bison or yak and non-ruminant species such as a donkey, horse, mule or pig.

[0055] As used herein, the term ‘genetic elements’ includes but is not limited to plasmids, promotors, terminators, coding sequences, cDNA, gDNA, RNA, enhancers, transcription factors and regulatory genes.

[0056] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0057] Although the present invention is broadly as defined above, those persons skilled in the art will appreciate that the invention is not limited thereto and that the invention also includes embodiments of which the following description gives examples.BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The invention will now be described with reference to the Figures in which:

[0059] Figure 1 shows colony morphology of various E. festucae strains which have transfected with ivrA and lovl, which encode a HMG-CoA reductase and an efflux pump, respectively, reducing sensitivity of E. festucae to lovastatin.

[0060] Figure 2 shows lovastatin production by E. festucae in culture. (A) Total ion count chromatogram of DCM extracts of wild-type and DHML producer YL316 1. (B) Total ion count and extracted ion chromatogram of lovastatin (M+H+ = 405.2) in strain YL316-329. My celia were cultured in CD YE medium for 5 days. (C) Schematic of the lovastatin biosynthetic pathway.

[0061] Figure 3 shows dynamics of the production of lovastatin by E. festucae in culture over time. (A) pH of growth media. (B) Lovastatin yield in CD YE media. (C) Lovastatin yield in PD media. Error bars represent standard error of 3 biological replicates.

[0062] Figure 4 shows the expression levels of lovBCGAFD genes in YL316-329 E. festucae strains in culture and in planta. A) Transcript levels of the lovBCGAFD genes in YL316-329 4, 5 and 15 in 5 d CD YE culture relative to the EF-2 and S22 housekeeping genes. (B) Transcript levels of lovBCGAFD in CD YE culture (“C”) and in planta 9 weeks post inoculation (“P”). Error bars represent standard error of 3 biological replicates.

[0063] Figure 5 shows metabolite profiles of YL316-329 strains in CD YE culture. Extracted ion counts of the corresponding metabolites as analysed by LCMS.

[0064] Figure 6 shows the cellular localisation of A. terreus lovastatin biosynthetic enzymes in E. festucae, viewed by fluorescence microscopy. The enzymes were expressed with a C- terminal GFP tag under the control of the constitutive elements Ptef / Ttub and observed under the fluorescence microscope after 7 days of growth on water agar. C: cytoplasm, V: vacuoles, ER: endoplasmic reticulum, PM: plasma membrane.

[0065] Figure 7 shows lovastatin production by E. festucae in planta. Extracted ion chromatogram of lovastatin (M+H+ = 405.2563) in the DCM extracts of plant tissue infected with wild-type, (A) YL316-329 and (B) SK259-265 strains of E. festucae.

[0066] Figure 8 shows in planta expression levels of lov and Itm genes in YL316-329 and SK259-265 E. festucae strains. Expression of (A) Itm genes and (B) lov genes in YL316-329 (P / c / A / PZ / ' C) and SK259-265 (P / / / 7?) strains in planta. (C) Expression of ectopically-integrated P Itm lov genes and in loco Itm genes in SK259-265. Error bars represent standard error from 3 biological replicates. * indicates p < 0.05 (two-tailed t-test).DETAILED DESCRIPTION OF THE INVENTION

[0067] The inventors have discovered a method for reducing the emission of methane from an animal. The method involves infecting a plant with an endophyte that has been genetically modified to produce a methane inhibitor.

[0068] The methane inhibitor is a compound that reduces the emission of methane from an animal when the compound is ingested by the animal. For example, a plant that is to be ingested by an animal (e.g. grass in a pasture containing livestock animals) may be infected with an endophyte modified to produce a methane inhibitor in planta. When the plant is ingested by the animal, the animal also ingests the methane inhibitor produced by the modified endophyte, which reduces the amount of methane emitted by the animal. For example, the methane inhibitor may be a methanogen inhibitor. Suitable methane inhibitors and their associated chemical classes include, but are not limited to, a statin (such as lovastatin), a haemanthamine, a bromoform, a bacteriocin, a non-ribosomal peptide, a flavonoid, a nitro compound, or any methane inhibitor that can be biochemically synthesised in a biological cell. In some embodiments, the methane inhibitor is a statin, a haemanthamine or a bromoform.

[0069] In one aspect, the present invention provides a process for preparing a modified endophyte capable of producing a statin, such as lovastatin, wherein the process comprises modifying an endophyte to insert genetic elements, wherein the genetic elements comprise genes for the selective biosynthesis of the statin in planta with a host plant, wherein the endophyte is E. festucae and the host plant is a grass, preferably a ryegrass.

[0070] The present invention relates to animals that emit methane, e.g., due to methanogenesis in the animal’s digestive tract. Such animals include livestock animals that are often reared in large numbers for agricultural purposes. Accordingly, one object of the invention is to provide means of reducing methane emissions from livestock animals and, therefore, reduce methane pollution associated with farming those livestock animals. The animal may be a ruminant or non-ruminant species. Ruminant species typically produce a greater amount of methane and, therefore, the invention is particularly advantageous when the animal is a ruminant species, such as a cattle, sheep, goat, deer, bison or yak.

[0071] The plant may be provided on a substate, e.g., an environment suitable for growing the plant to be ingested by the animal. Such substrates may include arable land for growing crops or pasture containing livestock animals, such as cows, sheep, goats, etc.

[0072] The modified endophyte is an organism that has been genetically modified to produce a one or more methane inhibitors. The modified endophyte may be a bacterial endophyte or a fungal endophyte. In some embodiments, the fungal endophyte is of the genus Epichloe, e.g. Epichloe festucae. The endophyte may be derived from a wild-type strain, e.g. a wild-type strain of Epichloe festucae (strain Fll). The wild-type strain may be engineered, e.g., to express lovastatin biosynthetic and resistance genes conferred by the DNA plasmids pYL316 and pYL329. Those persons skilled in the art will appreciate other strains, such as commercially available strains of E. festucae may also be useful in the invention.

[0073] The modified endophyte according to the present invention comprises one or more genetic elements. For example, the genetic elements may be selected from plasmids, promotors, terminators, cDNA, gDNA, RNA, enhancers, transcription factors, and regulatory genes. The primary purpose of the genetic elements is to enable the biosynthesis of the methane inhibitor. However, further genetic elements may be inserted into the modified endophyte, e.g., to confer resistance to the methane inhibitor and / or to encode for an efflux pump.

[0074] Genetic engineering of the endophyte may be performed by plasmid DNA transformation. The DNA transformation may include, e.g., introducing the relevant DNA plasmids into protoplasts of the endophyte, and isolating and propagating the transformants stably carrying and expressing the target genes. Protoplasts competent of taking up exogenous DNA may be prepared by digesting the cell walls of E. festucae mycelia with a combination of lysis enzymes. Preferably, the modified endophyte is prepared using MIDAS (as described is WO 2019 / 064242 Al).

[0075] The genetic elements comprise gene sequences for the biosynthesis of the methane inhibitor. For example, when the desired methane inhibitor is lovastatin, the genetic elements may comprise d, terreus genes selected from lovA, lovB, lovC, lovD, lovF, lovG or a combination of any two or more thereof. In some embodiments, the genetic elements may comprised, terreus genes lovB, lovC and lovG. In some embodiments, the genetic elements may comprised, terreus genes lovA, lovD and lovF. The genetic elements may comprise a heterologous promotor sequence and terminator sequence. For example, the biosynthesis genes may be placed under the control of a promoter sequence and a terminator sequence from Aspergillus nidulans, such as the Ptefl promoter sequence and TtubB terminator sequence from Aspergillus nidulans. Alternatively, the modified endophyte may comprise heterologous gene sequences for the biosynthesis of the methane inhibitor under the regulatory control ofendogenous promotor and terminator sequences. For example, when the endophyte is E. festucae, it is preferable to incorporate the gene sequences for the biosynthesis of the methane inhibitor, e.g. a statin such as lovastatin, under the regulatory control of Itm promotors and terminators. Advantageously, the biosynthetic pathway utilising the Itm promotors and terminators may provide high selectivity for in planta expression and / or high production of the methane inhibitor.

[0076] The genetic elements may be inserted into the endophyte by conventional techniques, e.g. as gDNA or cDNA sequences. However, the genetic elements are preferably inserted into the endophyte as cDNA sequences. The inventors have discovered that, surprisingly, inserting the gene coding sequences for the biosynthesis of the methane inhibitor as cDNA sequences may reduce issues such as missplicing. Those persons skilled in the art will appreciate that other genetic elements, as appropriate, may be inserted as cDNA.

[0077] In one aspect, the present invention provides a process for preparing a modified endophyte capable of producing a methane inhibitor, the process comprising modifying an endophyte to insert genetic elements, wherein the genetic elements comprise genes for the selective biosynthesis of the methane inhibitor in planta with a host plant, and wherein the genetic elements are inserted into the endophyte as cDNA.

[0078] The method for preparing a modified endophyte may integrate large genetic constructs into the E. festucae genome. For example, the genetic elements inserted into the endophyte may be between 5 kb and 60 kb, e.g., between 10 kb and 60 kb, 15 kb and 60 kb, 20 kb and 60 kb, 25 kb and 60 kb, 30 kb and 60 kb, 35 kb and 60 kb, 40 kb and 60 kb, 45 kb and 60 kb, 50 kb and 60 kb, or 55 kb and 60 kb. In some embodiments, the genetic elements inserted into the endophyte are at least 1 kb, e.g., at least 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, 50 kb, 55 kb or 60 kb. In some embodiments, the genetic elements inserted into the endophyte are at least 40 kb, 45 kb, 50 kb, 55 kb or 60 kb. Surprisingly, the large genetic constructs may be integrated without negatively impacting the endophyte-ryegrass symbiosis or phenotypes of the endophyte or ryegrass. The inventors observed no statistically significant differences between ryegrass infection rates of genetically modified E. festucae strains according to the present invention and unmodified parent strains. Additionally, no differences were observed in the gross plant morphology between plants infected with modified strains according to the present invention.

[0079] The genetic elements may be inserted into the endophyte as a single genetic construct or as multiple constructs. In one aspect, the present invention provides a process for preparing a modified endophyte which randomly integrates heterologous biosynthetic pathways either as a single genetic construct or as multiple constructs into the endophyte. Surprising, heterologous biosynthetic pathways may be randomly integrated either as a single genetic construct or as multiple constructs into the E. festucae genome without negatively impacting the endophyteryegrass symbiosis or phenotypes of the endophyte or ryegrass.

[0080] The modified endophyte has a symbiotic relationship when grown in planta with the host plant. It may contain genes which only produce the desired methane inhibitor when the endophyte is in planta. By taking advantage of genetic elements which are predominantly active in planta, heterologous biosynthesis can be controlled in a targeted manner i.e production of the methane inhibitor can be restricted to when the modified endophyte is in planta (selectively with the host plant). Accordingly, an advantage of the present invention is providing a modified endophyte that selectively expresses the methane inhibitor in planta with the host plant. Preferably, nil or a negligible amount of the methane inhibitor is expressed when the endophyte is growing epiphytically, e.g., ex planta or when the endophyte is exposed to a plant besides the host plant. Advantageously, selective activation of the endophyte in planta avoids or minimises undesirable expression of the methane inhibitor biosynthesis genes or production of the methane inhibitor outside of the target environment. For example, in some embodiments the host plant is a ryegrass and the modified endophyte selectively expresses the methane inhibitor, e.g. a statin such as lovastatin, in planta when the ryegrass is infected with the modified endophyte.

[0081] The intra- or extracellular localisation of biosynthesised compounds, such as lovastatin, can be highly variable depending on the fungal species and growth medium (Srinivasan et al., 2022). Preferably, the modified endophyte according to the present invention expresses the methane inhibitor extracellularly. For example, the inventors have developed a modified E. festucae in which lovastatin expression was localised almost exclusively in the extracellular fraction when the E. festucae were cultured in either CD YE or PD media. It is believed that extracellular expression of the methane inhibitor is advantageous for secreting the methane inhibitor to the plant environment. Additionally, it is believed extracellular expression of the methane inhibitor reduces harmful effects due to the methane inhibitor toxicity to the endophyte. Without wishing to be bound by theory, it is believed the incorporation of an efflux pump into the modified endophyte may favour extracellular localisation of the methane inhibitor.

[0082] Problems can arise with heterologous biosynthesis, such as toxicity or sensitivity of the host to the genetic product. This can be overcome via insertion of genetic elements into the host which, when transcribed, confer reduced sensitivity or increased tolerance to the foreign product. For example, to combat the toxic effects of lovastatin on A. festucae, resistance genes encoded within the A. terreus lovastatin cluster were inserted into E. festucae, which encode a lovastatin-resistant HMG-CoA reductase (ivrA) and an efflux pump (lovl). This leads to expression of efflux pumps in the host capable of transporting the lovastatin out of the intracellular space, and providing an active HMG-Co-A reductase to confer resistance to the reductase inhibition activities of lovastatin. This aids in allowing creation of a successful heterologous biosynthetic production system, with an increased output which is not limited by the innate lovastatin sensitivity of the endophyte or host plant. Accordingly, in some embodiments, the modified endophyte may further comprise genetic elements which confer resistance to the methane inhibitor (i.e. genes that provide resistance to the methane inhibitor), genetic elements that encode an efflux pump, or both.

[0083] The host plant to be infected can be any suitable pasture plant which is capable of forming a symbiotic relationship with endophytes. Suitable plants include, but are not limited to, the genera I.olium. Festuca, Poa, Daclylis. Agrostis and Phleum. In one aspect, the invention provides a plant infected with a modified endophyte, wherein the modified endophyte produces a methane inhibitor in planta. As described above, preferably the modified endophyte selectively expresses the methane inhibitor within the host plant.

[0084] In one aspect, the present invention provides a process for preparing a modified endophyte capable of producing a methane inhibitor, wherein the process comprises modifying an endophyte to insert genetic elements, wherein the genetic elements comprise genes for the selective biosynthesis of the methane inhibitor in planta with a host plant, wherein the endophyte is E. festucae and the host plant is a grass, preferably a ryegrass.

[0085] The step of infecting the plant as described herein encompasses infecting the host plant in planta and infecting a precursor of the host plant, e.g. a seed or seedling.

[0086] Advantageously, the present invention provides a method for both producing a methane inhibitor and delivering the methane inhibitor to an animal. The process comprises providing a host plant infected with a modified endophyte that is capable of producing the methane inhibitor such that the methane inhibitor is produced in planta. The infected plant maybe provided by infecting the host plant in situ. Alternatively, the infected plant may be provided by growing the host plant from an infected seed.

[0087] The methane inhibitor is then delivered to the animal by feeding the infected plant to the animal. The plant may be fed to the animal by grazing the animal on the plant on the substrate. For example, livestock may be grazed on pastures with a plant, e.g. a grass, infected with the modified endophyte. Alternatively, the plant may be fed to the animal by harvesting the plant and feeding the harvested plant to the animal. For example, a suitable plant, such as a grass, may be infected with the modified endophyte. The plant is then harvested, and optionally further processed, and provided as a plant product such as silage, hay, straw or pellets suitable for use as an animal feed product. Those persons skilled in the art will appreciate the process may be used to prepare other forms of animal feed comprising a methane inhibitor provided that the animal feed comprises a plant suitable for infection with the modified endophyte. The modified endophyte may be supplied in the form of an infected seed, e.g., a grass seed containing the modified endophyte. The infected seed may be sowed in a suitable environment to produce a plant carrying the modified endophyte. The infected seed may be produced by allowing a parental infected plant to flower and produce seeds. Accordingly, in one aspect, the invention provides a seed infected with a modified endophyte, wherein the modified endophyte produces a methane inhibitor in planta. In some embodiments, the seed is a ryegrass seed infected with an E. festucae endophyte strain modified to produce a methane inhibitor in planta.Advantageously, the endophyte may be maintained continuously in a plant grown from infected seed, this allows for continuous delivery of the methane inhibitor.

[0088] Alternatively, the modified endophyte may be supplied as a culture. This culture may be used to infect a seed or plant as described above. For example, the modified endophyte may be supplied as a mycelium culture, e.g., to be infected into ryegrass. Advantageously, due to the selective expression described above, the methane inhibitor is only produced once the modified endophyte is in planta.

[0089] The following non-limiting examples are provided to illustrate the present invention and in no way limit the scope thereof.EXAMPLESExample 1Strains, growth conditions, and microscopy

[0090] Bacterial strains, fungal strains, plasmids and plant material used in this study are listed in Table S3. E.festucae strains were grown at 22°C on 2.4% (w / v) potato dextrose agar or broth or CD YE medium with shaking at 200 r.p.m. E.festucae protoplasts were prepared and transformed as previously described (Itoh et al., 1994; Young et al., 2005). For fluorescence microscopy, E.festucae hyphae were cultured on 1.5% agar for 7 days and imaged using a BX63 fluorescence microscope (Olympus).Plant manipulations

[0091] L. perenne seeds were surface sterilised using a method adapted from Latch and Christensen (1985). Seeds were soaked in 50% (v / v) H2SO4 for 30 min, rinsed in sterile H2O and soaked in 50% (v / v) commercial bleach (NaOCl 21.5 g / L), rinsed thoroughly with sterile H2O and air-dried in a laminar flow cabinet. L. perenne seedlings (7 d old germinated on 1.5% agar) were inoculated using a method adapted from Latch and Christensen (1985). Briefly, a shallow 2-3 mm long incision was made longitudinally between the mesocotyl and coleoptile regions of the seedling and inoculated by placing a small amount of Epichloe mycelia into this cut.Inoculated seedlings were then maintained on agar for 7 days in darkness followed by 7 days with a 16 h photoperiod, then were transferred to root trainers containing fungicide-free organic seed mix (Daltons®, New Zealand) and maintained in a Conviron GEN1000 plant growth chamber with a photoperiod of 16 h of light (25% intensity) and 75% humidity.DNA extraction and PCR screening for endophyte infection

[0092] Plant tissues were lysed with the MPBio FastPrep24 5G bead beater in lysis buffer (400 mM Tris-HCl pH 8, 60 mM EDTA, 150 mM NaCl) (Liu et al., 2000). SDS was added to a concentration of 1% and mixed by inversion, followed by addition of precipitation solution (3 M potassium acetate, 10% glacial acetic acid, pH 4.8) and centrifugation at 17,000 x g for 5 min. An equal volume of isopropanol was added to the supernatant and DNA was precipitated by centrifugation, washed with 75% (v / v) ethanol and resuspended in 100 pL Tris-EDTA buffer. 1 pL of DNA was used as template for multiplex PCR reactions using primers YL705 / YL706 (E. festucae ribosomal protein S22; 671 bp product size) and YL501F / YL501R (L. perennecinnamoyl-CoA reductase 1; 112 bp product size) using the OneTaq® Quick-Load® 2X Master Mix with Standard Buffer (NEB).Metabolite extraction and mass spectrometry

[0093] Five-day old mycelial cultures (25 ml) were freeze-dried and resuspended in 5 ml 1% HC1. Freeze-dried plant pseudostem samples (50-200 mg dry weight) were ground into fine powder using MPBio FastPrep24 5G bead beater grinder and lysis system (40 s, 6m / s) and resuspended in 1 ml 1% HC1. Metabolites were extracted with 3 ml (for 25 ml mycelial cultures) or 0.5 ml (for plant pseudostem samples) dichloromethane (DCM) and resuspended in acetonitrile (MeCN). Liquid Chromatography-Mass Spectrometry (LC-MS) was performed on an Agilent 1260 Infinity II LC-MS system with DAD and electrospray ionisation. A Phenomenex C 18 Kinetex column (2.6 p, 100 A, 50 * 2.1 mm) equipped with a Phenom enex Cl 8 guard cartridge and maintained at 40°C was eluted with a mobile phase of A: H2O and B: MeCN, both containing 0.1% formic acid. An injection volume of 10 pL and flow rate of 0.3 mL / min were used. The gradient was TO 20% B, T1 20% B, T24 90% B, T25 100% B, and T30 100% B, followed by a 3 min equilibration back to the initial conditions. Sample lovastatin concentrations were calculated from standard curves generated using lovastatin standards (Sigma-Aldrich) ranging in concentration from 1 ng / ml to 10 pg / ml.

[0094] High-resolution mass spectrometric data and tandem mass spectra were obtained with an Agilent 6530 Q-TOF fitted with an electrospray ion source and equipped with an Agilent 1260 Infinity II LC system. Chromatography was carried out using an Agilent Accucore C18 2.0 pm 50 x 2.1 mm column eluted with a mobile phase of A: H2O and B: MeCN, both containing 0.05% formic acid. The flow rate was 0.3 mL / min and injection volume 5 pL. The gradient used was TO 20% B, T1 20% B, T24 100% B, and T29 100% B, with equilibration to the initial conditions. The MS parameters used were: positive ion mode, capture window of 100-1000 m / z, acquisition rate 2 scans / s, capillary temperature 300°C, capillary voltage 3500 V, fragmentor voltage 175 V, drying gas flow 8 L / min, sheath gas temp 350°C, sheath gas flow 11 L / min, and a nebulizer pressure of 35 psi. MS / MS data were acquired for selected masses using collision- induced dissociation with an isolation window of 1.6 and collision energy of 10 eV.RNA isolation, reverse transcription and quantitative PCR

[0095] Plant tissues or mycelia were homogenised in TRIzol using 1 mm glass beads in a bead beater (FastPrep24, MPBio) and RNA was isolated using the PureLink Plant RNA Kit(Invitrogen) according to the manufacturer’s instructions. cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen) and qPCR was performed using SsoFast Evagreen supermix (Bio-Rad) with 50 pM ROX (Invitrogen) using a QuantStudio 3 Real-Time PCR System (Applied Biosystems) with two technical replicates per sample. Standard curves were generated for each primer set using 1 to 1,000,000 copies of template. Target transcript levels were normalised against the E. festucae elongation factor 2 (EF-2; EfM3.021210) and ribosomal protein S22 (EfM3.016650) reference genes (Chujo & Scott, 2014).Example 2: Engineering biosynthesis of lovastatin in Epichloe festucae Ell - mitigation of lovastatin sensitivity and incorrect intron splicing

[0096] For the initial generation of a lovastatin-producing strains of E. festucae, two multigene expression plasmids were prepared using MIDAS (van Dolleweerd et al., 2018). The genomic sequences of A. terreus genes lovB / C / G were assembled to create plasmid pSK126, while genes lovA / F / D were assembled to create plasmid pSK148. Regulatory promoter and terminator sequences for these genes were derived from the PAX biosynthetic gene cluster (BGC) of Penicillium paxilli and the JAN BGC of Penicillium janthinellum, which had been previously validated for heterologous expression of secondary metabolites in other filamentous fungi. These two plasmids were co-transformed into E. festucae, but neither lovastatin nor its intermediates were detected in chemical analyses of the resulting transformants grown in liquid cultures. After validating expression of the LOV genes by RT-qPCR, other potential issues including missplicing, enzyme mislocalisation and lovastatin toxicity were investigated. RNAseq of the SKI 26- 148 strains revealed that majority of the terreus lovBCAF transcripts were misspliced in E. festucae. To overcome intron missplicing, cDNA sequences of the genes were used for subsequent experiments.

[0097] Toxicity assays revealed sensitivity of wild-type E. festucae strain Fll to lovastatin at concentrations >5 pg / ml (Figure 1). To mitigate this, two resistance genes encoded within the A. terreus lovastatin cluster were introduced into E. festucae that encode a lovastatin-resistant HMG-CoA reductase (ivrA) and an efflux pump (lovl) (Itoh et al., 2018; Ley et al., 2015). Strains transformed with a plasmid containing both of these resistance genes (pSK256) were found to be more tolerant to lovastatin than the wild-type (Figure 1), with growth observed on media containing up to 25 pg / ml lovastatin. As such, ivrA and lovl were included in the generation of all subsequent strains.Example 3: In culture production of DHML and lovastatin by E. festucae

[0098] A multigene expression plasmid (pYL316) was prepared by assembling the cDNA sequences of A. terreus genes lovB, lovC, and lovG, with each gene placed under the control of the constitutively active Vtefl promoter sequence and LtubB terminator sequence from Aspergillus nidulans (Lukito et al., 2019). This plasmid was transformed into protoplasts wildtype E. festucae strain Fl 1. Growth of the resulting strains in liquid CD YE medium for five days resulted in the detection of dihydromonacolin L (DHML; the expected pathway product of LovB, LovC, and LovG) in extracts from nine out of 16 transformants (Figure 2A). The strain that produced the most DHML (YL316 1) was subsequently transformed with a second plasmid (pYL329) that contained cDNAs of the A. terreus genes lovA, lovF, and lovD, which encode proteins that convert DHML into lovastatin via monacolin L and monacolin J (Figure 2C). Each of these genes were placed under the regulatory control of the constitutively active promoter sequence and terminator sequence of the trpC gene from A. nidulans (PlrpC and LtrpC). Lovastatin and monacolin J were detected in culture extracts from 11 out of the resulting 15 YL316_l ::pYL329 transformants (Figure 2B), demonstrating that no additional modifications are required to engineer lovastatin biosynthesis into E. festucae.

[0099] The intra- or extracellular localisation of lovastatin can be highly variable depending on the fungal species and growth medium (Srinivasan et al., 2022). As it was not known if lovastatin was intra- or extracellular in E. festucae, lovastatin extraction was performed on entire cultures up to this point. The lovastatin yield of the best producing strain (YL316-329 4) grown under these conditions was approx. 0.3 mg / kg mycelium (dry weight) or 5 pg per litre of culture. We next investigated the dynamics of lovastatin production in A. festucae YL316-329 4 in CD YE and PD media over 7 days. We found that both CD YE and PD media resulted in comparable lovastatin yields, and lovastatin was localised almost exclusively in the extracellular fraction in both media types and across all time points (Figure 3).Example 4: Correlation of gene transcript levels with production of pathway products

[0100] Quantitative reverse transcription PCR (RT-qPCR) analysis was performed for all of the lovastatin biosynthesis genes from plasmids pYL316 and pYL329 using RNA templates extracted from three independent lovastatin-producing E. festucae strains cultured in CD YE medium for 5 days. The results showed that the LOV genes were expressed at approximately 1.5% of the transcript levels of EF-2 and S22 housekeeping genes (Figure 4A). Interestingly, expression of the lovD gene, which encodes the acyl transferase that catalyses the final reactionto synthesise lovastatin from monacolin J, was lower than all the other five LOV genes in all three strains analysed. A marked accumulation of monacolin J was correspondingly observed in the YL316-329 strains (Figure 5), suggesting that increasing lovD gene expression might be an immediate functional strategy for increasing lovastatin production in this strain.Example 5: Localisation of lovastatin biosynthetic enzymes in E. festucae

[0101] To our knowledge no localisation studies have been performed on the lovastatin biosynthetic enzymes in any fungus. As lovastatin biosynthesis may be confined to subcellular compartments, we analysed the cellular localisation of the LOV enzymes by expressing C- terminal GFP-fused versions of the enzymes in E. festucae . As shown in Figure 6, fluorescence microscopy of the resulting strains suggested that LovA-GFP localised to the endoplasmic reticulum, as would be expected for a cytochrome P450 monooxygenase; the efflux pump Lovl- GFP localised to both plasma membrane and vacuoles, as reported previously for a homologous efflux pump (Ley et al., 2015); LovB-GFP localised to both cytosol and vacuoles, as reported for other polyketide synthases (Pandith et al., 2019); and LovC-GFP, LovG-GFP, and LovD-GFP all localised to the cytosol (Figure 6). Taken together, these findings do not suggest significant enzyme mislocalisation issues in E. festucae. The localisation of the efflux pump LovI to the vacuoles as well as the cell membrane suggests that lovastatin could be transported both into the vacuole as well as into the periplasmic space.Example 6: In planta production of lovastatin by E. festucae

[0102] The lovastatin-producing E. festucae strains YL316-YL329 4 and YL316-YL329 5, and wild-type E. festucae as control, were subsequently inoculated into perennial ryegrass and the infection status of the plants were determined at eight-weeks post-inoculation by PCR. No statistically significant differences between infection rates of the two strains were observed (Table 1) and no differences were observed in the gross plant morphology between plants infected with the two strains. HPLC-ESI-MS / MS analysis of DCM extracts of pseudostem tillers confirmed the presence of lovastatin in plant tissues infected with either strain (Figure 7). The highest observed titre was 0.025 mg / kg d.w. plant tissue, equivalent to approximately 9% of the culture titre of the YL316-329 4 strain (0.285 mg / kg). RT-qPCR analysis showed that all six lovBCGAFD genes were indeed more highly expressed in the plant (median fold difference of 9.2) as compared to 5 day old axenic cultures grown in CD YE media for both YL316-329 strains (Figure 4B), potentially accounting for the higher lovastatin titre of the strains per unit of fungal biomass in planta.Table 1No. infected No. uninfected Infection rate -value (chi-squared test)Wild-type H 23 32% -YL316-329 4 4 28 12.5% 0.054YL316-329 15 932% 0.986

[0103] To test an alternative regulatory system for the production of lovastatin in planta, the genes for lovastatin biosynthesis were placed under the control of the LTM regulatory elements. The Itm genes encode enzymes for the production of the secondary metabolite lolitrem B and related indole diterpenes and are some of the most highly expressed genes when E.festucae is in planta, but are transcriptionally silent in culture (Fleetwood et al., 2007; Tanaka et al., 2005; Young et al., 2006; Zhang et al., 2009). New genetic constructs (pSK259 and pSK265) were prepared with the lov genes under the regulatory control of Itm promoters and terminators and transformed into wild-type E. festucae, with the resulting transformants inoculated into perennial ryegrass. Lovastatin was extracted from pseudostems of infected plants as well as from in vitro cultures of SK259-265. As expected, negligible amounts of lovastatin were detected via high resolution LCMS in the SK259-265 strains grown in culture, suggesting that the lov genes were expressed at very low levels in axenic culture. This result was consistent with the previously demonstrated in planta-specific activation of the Itm genes (Young et al., 2005), which are only expressed during endophytic growth of the fungi (Chujo & Scott, 2014). However, lovastatin was produced when these strains were grown in planta, at a maximum observed titre of 0.013 mg / kg d.w. (Figure 7).

[0104] As expected, RT-qPCR analysis showed no significant differences in the expression of the endogenous Itm genes between the Pltm-regulated strains (SK259-265) and the WefA / VtrpC- regulated strains (YL316-329) in the plant (Figure 8A). However, in planta expression of the five lovastatin biosynthetic genes lovBCGAF were higher in SK259-265 than in YL316-329 (Figure 8B). Expression of the final gene, lovD, was however considerably lower than the other five lov genes, and not differentially expressed between the two E. festucae strains (Figure 8B), a phenomenon similarly observed in the YL316-329 strains in culture. The expression of lovD potentially plays a role in the low lovastatin titre of the SK259-265 strains despite higher lovAFCBG gene expression. However, unlike the YL316-329 in culture, there was no evidence of monacolin J accumulation in the SK259-265 strain in planta.

[0105] Finally, the presence of in loco (the endogenous Itm genes) and ex loco (the lov genes which are randomly and thus likely ectopically-integrated) in the SK259-265 strains allowed us to test for locus-dependent effects or requirements of the Itm promoters, as these gene “sets” are under the regulation of the same Itm promoters, but one is in loco and another ex loco. RT-qPCR analysis showed four out of the six gene “sets” exhibit comparable gene expression (Figure 8C), while mixed results were observed with the two other gene sets, in which one showed higher expression of the in loco gene (genes under PltmS), while another showed higher expression of the ectopic construct (genes under PltmP) (Figure 8C). Taken together, this analysis does not suggest that in loco integration of the Itm regulatory elements into the subtelomeric LTM locus is required for achieving levels of gene expression necessary for the heterologous production of secondary metabolites.

[0106] It is not the intention to limit the scope of the invention to the abovementioned examples only. As would be appreciated by a skilled person in the art, many variations are possible without departing from the scope of the invention as set out in the accompanying claims.

Claims

CLAIMS1. A method for preparing a modified endophyte capable of producing a methane inhibitor, wherein the method comprises the step of inserting genetic elements into an endophyte to provide the modified endophyte, wherein the genetic elements comprise gene coding sequences, promoters, terminators, and other regulatory elements for selective biosynthesis of the methane inhibitor in planta within a host plant.

2. The method of claim 1, wherein the genetic elements are inserted into the endophyte as cDNA.

3. The method of claim 1 or 2, wherein the genetic elements inserted into the endophyte are between 1 kb and 60 kb.

4. The method of any one of claims 1 to 3, wherein the endophyte is modified using MIDAS.

5. A modified endophyte capable of producing a methane inhibitor, wherein the modified endophyte comprises genetic elements comprising gene coding sequences, promoters, terminators, and other regulatory elements for selective biosynthesis of the methane inhibitor in planta within a host plant.

6. The method of to any one of claims 1 to 4 or the modified endophyte of claim 5, wherein the modified endophyte is a modified bacterial endophyte or a modified fungal endophyte.

7. The method of any one of claims 1 to 4 and 6 or the modified endophyte of claim 5 or 6, wherein the modified fungal endophyte belongs to the genus Epichloe; optionally wherein the modified fungal endophyte is Epichloe festucae.

8. The method of any one of claims 1 to 4, 6 and 7 or the modified endophyte of any one of claims 5 to 7, wherein the genetic elements comprise gene coding sequences for biosynthesis of the methane inhibitor.

9. The method of any one of claims 1 to 4 and 6 to 8 or the modified endophyte of any one of claims 5 to 8, wherein the genetic elements comprise gene coding sequences amplified from biological material or produced synthetically for biosynthesis of the methane inhibitor, andwherein the genetic elements comprise promoter and terminator sequences selective for in planta expression of the methane inhibitor.

10. The method of any one of claims 1 to 4 and 6 to 9 or the modified endophyte of any one of claims 5 to 9, wherein the genetic elements comprise promoter and terminator sequences, amplified from the host endophyte, other biological sources, or produced synthetically, and wherein the genetic elements are selective for in planta expression of the methane inhibitor.

11. The method of any one of claims 1 to 4 and 6 to 10 or the modified endophyte of any one of claims 5 to 10, wherein the heterologous gene sequences for biosynthesis of the methane inhibitor are under regulatory control of endogenous promotors and terminators.

12. The method of any one of claims 1 to 4 and 6 to 11 or the modified endophyte of any one of claims 5 to 11, wherein the heterologous gene sequences for biosynthesis of the methane inhibitor are under regulatory control of Itm promotors and terminators, optionally wherein the heterologous gene sequences for biosynthesis of the methane inhibitor are lov gene sequences under regulatory control of Itm promotors and terminators.

13. The method of any one of claims 1 to 4 and 6 to 12 or the modified endophyte of any one of claims 5 to 12, wherein, the modified endophyte comprises an Itm promotor and an Itm terminator.

14. The method of any one of claims 1 to 4 and 6 to 10 or the modified endophyte of any one of claims 5 to 10, wherein the genetic elements comprise gene sequences with heterologous promoters and terminators.

15. The method or the modified endophyte of claim 14, wherein the genetic elements comprise a promoter sequence and a terminator sequence from Aspergillus nidulans. preferably, the genetic elements comprise Ptefl promoter sequence and a TtubB terminator sequence from Aspergillus nidulans.

16. The method of any one of claims 1 to 4 and 6 to 15 or the modified endophyte of any one of claims 5 to 15, wherein the gene sequences for biosynthesis of the methane inhibitor are lov gene sequences e.g. lovA, lovB, lovC, lovD, lovF, lovG or a combination of any two or more thereof, preferably, lovB, lovC, and lovG or lovA, lovD and lovF.

17. The method of any one of claims 1 to 4 and 6 to 16 or the modified endophyte of any one of claims 5 to 16, wherein the modified endophyte produces the methane inhibitor in planta at a rate of at least 0.005 mg / kg host plant (dry weight), preferably at a rate of at least 0.01 mg / kg host plant (dry weight).

18. The method of any one of claims 1 to 4 and 6 to 17 or the modified endophyte of any one of claims 5 to 17, wherein the endophyte is modified to reduce toxicity caused by the methane inhibitor.

19. The method of any one of claims 1 to 4 and 6 to 18 or the modified endophyte of any one of claims 5 to 18, wherein the genetic elements comprise:• a gene sequence for biosynthesis of the methane inhibitor, under regulatory control of endogenous promotors and terminators, such as Itm promotors and terminators in E. feslucae:• a gene sequence encoding an efflux pump;• a gene sequence that confers resistance to the methane inhibitor, such as a heterologous gene sequence that encodes a reductase that confers resistance to the methane inhibitor.

20. The method of any one of claims 1 to 4 and 6 to 19 or the modified endophyte of any one of claims 5 to 19, wherein the genetic elements enable extracellular localisation of the methane inhibitor.

21. The method of any one of claims 1 to 4 and 6 to 20 or the modified endophyte of any one of claims 5 to 21, wherein the methane inhibitor is a methanogen inhibitor, optionally the methane inhibitor is any one of a statin, a haemanthamine, a bromoform, a bacteriocin, a non- ribosomal peptide, a flavonoid, a nitro compound, or any methane inhibitor or precursor thereof that can be biosynthesised in a biological cell.

22. The method of any one of claims 1 to 4 and 6 to 21 or the modified endophyte of any one of claims 5 to 21, wherein the methane inhibitor is lovastatin.

23. The method of any one of claims 1 to 4 and 6 to 22 or the modified endophyte of any one of claims 5 to 22, wherein the host plant is a grass, preferably, the grass belongs to any one of the genera Lolium, Festuca, Poa, Dactylis, Agrostis and Phleum.

24. The method of any one of claims 1 to 4 and 6 to 23 or the modified endophyte of any one of claims 5 to 23, wherein the host plant is a ryegrass.

25. The modified endophyte of any one of claims 5 to 24, wherein the endophyte is an isolated cell.

26. A method of delivering a methane inhibitor to a host plant, the method comprising infecting the host plant, or a precursor thereof, with a modified endophyte of any one of claims 5 to 25.

27. A method for reducing methane emissions from an animal, the method comprising: al. providing a host plant infected with a modified endophyte of any one of claims 5 to 25, wherein the modified endophyte produces the methane inhibitor in planter, bl. feeding the plant comprising the methane inhibitor to the animal; or the method comprising: a2. growing a host plant on a substrate, wherein the host plant is infected with a modified endophyte capable of producing a methane inhibitor and the modified endophyte produces the methane inhibitor in plan la b2. allowing the animal to graze on the plant on the substrate.

28. The method of claim 27, wherein step (bl) comprises harvesting the host plant and feeding the harvested plant to the animal.

29. The method of claims 27, wherein step (bl) or (b2) comprises grazing the animal on the host plant, e.g., wherein the host plant is on a substrate, e.g. wherein the substrate is pasture land or arable soil.

30. The method of any one of claims 27 to 29, wherein the animal is a livestock animal; optionally, wherein the livestock animal is a cattle, sheep, goat, deer, donkey, horse, bison, yak or mule.

31. The method of any one of claims 27 to 30, wherein the animal is a ruminant, preferably, the ruminant is a cattle, sheep, goat, deer, bison or yak.

32. A plant, or precursor thereof, comprising a modified endophyte according to any one of claims 5 to 25, wherein the precursor thereof is a seed or seedling.

33. A seed or seedling infected with a modified endophyte of any one of claims 5 to 25, wherein the seed is a ryegrass seed infected with the modified endophyte.

34. A culture comprising a modified endophyte of any one of claims 5 to 25.

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