Technical Field

[001] The technology relates to recombinant yeast expressing a vanadate-dependent bromoperoxidase for the production of bromoform. In particular the recombinant yeast can be used to reduce the formation of methane in animal digestive tracts.

Cross Reference to Related Applications

[002] This application claims priority to Australian provisional patent application number 2019901992 filed 7 June 2019, and Australian provisional patent application number 2019903125 filed 27 August 2019, which are herein incorporated by reference in their entireties.

Background

[003] Methane is a greenhouse gas produced primarily by methanogenic microbes, particularly methanogenic archaea. These microbes are present in the environment but also inhabit the gastrointestinal tract of ruminants and methane from farmed ruminants accounts for approximately 13% of all greenhouse gas emissions.

[004] The production of methane is known to reduce the partial pressure of hydrogen, which would otherwise inhibit fermentation in the rumen. However, methane in the rumen reduces the formation of volatile fatty acids which are crucial ruminant nutrition. Therefore inhibiting methane production is advantageous for improving animal feed conversion efficiency. Inhibition of methane production has been shown to increase feed conversion efficiency by up to 10%.

[005] Red macroalgae (seaweeds) can decrease methane emissions from ruminant organisms such as cattle when they are included in animal feed. This activity is due to the presence of brominated organic hydrocarbons, such as bromoform (CHBrs), which is predominant brominated hydrocarbon in seaweed. Brominated hydrocarbons prevent methane emission from ruminants by interfering with the vitamin B12 cofactor which is used by the methyltransferase enzymes of ruminant methangenic archaea in the production of methane.

[006] Bromoform is produced in seaweed by the action of vanadate-dependent bromoperoxidases which catalyse the halogenation of organic substrates in the presence of hydrogen peroxide, according to the reaction: RH + HBr + H2O2 RBr + 2 H2O, where RH is the organic substrate. [007] Bromoform-producing seaweeds such as Asparagopsis taxiformis grow relatively slowly in deep seawater. The large-scale cultivation of these seaweeds is therefore too slow and costly to satisfy the demand from animal agriculture. Alternatively, production of sufficient quantities seaweed in aquaculture systems is not presently viable.

[008] In contrast, technology for the large-scale cultivation and distribution of yeast cells or as animal feed is well established. However, the production of high levels of bromoform in cultured organisms has not been successful as bromoform is toxic and its production requires the one carbon organic substrates and hydrogen peroxide which are also toxic and cause cellular damage. Accordingly, it has not been possible to express a vanadate- dependent bromoperoxidases in a recombinant microorganism and use that organism to produce bromoform in sufficient concentrations to be useful, for example as a stock feed additive.

[009] The present inventors have developed a system to express functional vanadate- dependent bromoperoxidase in the peroxisome. These yeast produce bromoform at a concentration that allows the bromoform containing yeast to be used as a stock feed additive.

Summary

[010] In a first aspect, there is provided a recombinant yeast capable of producing bromoform, the recombinant yeast comprising at least one copy of an expression cassette comprising a constitutive or inducible promoter operatively coupled to a nucleic acid sequence encoding a vanadate-dependent bromoperoxidase (vBPO).

[011] The recombinant yeast may further comprise either or both of a nucleic acid sequence encoding a peroxisomal targeting signal; and a transcription termination sequence, wherein the coding sequence of the nucleic acid sequence encoding the peroxisomal targeting signal is in frame with the nucleic acid sequence encoding the vBPO.

[012] In one embodiment the vBPO is Alteromonas naphthalenivorans vBPO (SEQ ID NO: 3), Chondrus crispus 615 vBPO (SEQ ID NO: 4), Ascophyllum nodosum vBPO (SEQ ID NO: 5), Corallina officianlis vBPO (SEQ ID NO: 6), Corallina pilulifera (SEQ ID NO: 7), Gracilaria changii vBPO (SEQ ID NO: 8), Laminaria digitate vBPO (SEQ ID NO: 9), or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to any one of SEQ ID NOs: 3-9.

[013] In one embodiment the vBPO is G. changii vBPO (SEQ ID NO: 8) or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 8. [014] In one embodiment the vBPO is monomeric. For example vBPO may be A. marina vBPO (SEQ ID NO: 2) or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical toidentical to SEQ ID NO: 2.

[015] The amino acid sequence of the peroxisomal targeting signal may comprise any one of SEQ ID NOs: 10-14 or 21-27.

[016] In one embodiment the amino acid sequence of the peroxisomal targeting signal comprises the sequence PEALIKSMTSKL (SEQ ID NO: 14).

[017] The peroxisomal targeting signal may be C-terminal to the vBPO.

[018] In some embodiments the inducible promoter is a methanol inducible promoter, such as one of the AOX1 , AOX2, CTA1 , DAS1 , DAS2, FLD, and PMP20 promoters. In one the methanol inducible promoter is the AOX1 promoter, for example comprising SEQ ID NO: 1 or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical toto SEQ ID NO: 1.

[019] In some embodiments the expression cassette is present in a plasmid vector. In other embodiments the expression cassette is stably integrated into the yeast genome.

[020] In some embodiments the yeast is catalase deficient.

[021] The yeast may be selected from the group of genera consisting of Arxula, Candida, Ogataea, Kluyveromyces, Pichia, Saccharomyces, and Yarrowia

[022] In some embodiments the yeast is Pichia pasto s.

[023] In some embodiments the yeast is Saccharomyces cerevisiae.

[024] In a second aspect there is provided a method for producing bromoform containing yeast comprising

(a) culturing the yeast of the first aspect in the absence of methanol;

(b) adding methanol to the culture to induce the expression of the vBPO;

(c) further culturing the yeast under conditions suitable for the production of bromoform.

[025] The expressed vBPO may be targeted to the yeast peroxisome. In this embodiment the bromoform is produced in the yeast peroxisome.

[026] The method may further comprise harvesting the bromoform containing yeast.

[027] In some embodiments, in steps (a) and/or (c) the yeast is cultured in the presence of vanadium, preferably sodium orthovanadate. [028] Alternatively or in addition in steps (a) and/or (c) the yeast is cultured in the presence of a bromide, preferably sodium bromide or potassium bromide.

[029] In a third aspect there is provided the yeast produced by the method of the second aspect.

[030] In a fourth aspect there is provided an extract or concentrate of the yeast of the third aspect.

[031] In a fifth aspect there is provided an animal feed supplement comprising the yeast of the third aspect or the extract or concentrate of the fourth aspect.

[032] In a sixth aspect there is provided an animal feed comprising the yeast of the third aspect or the extract or concentrate of the fourth aspect.

[033] In a seventh aspect there is provided a method for reducing methane production in an animal comprising administering to the animal an effective amount of the yeast of the third aspect, the extract or concentrate of the fourth aspect, the feed supplement of the fifth aspect or the animal feed of the sixth aspect.

[034] The animal may be a ruminant or non-ruminant animal.

[035] The ruminant animal may be a bovine, ovine, caprine, equine, cervid, or camelid.

[036] The non-ruminant animal may be porcine or avian.

Definitions

[037] Throughout this specification, unless the context clearly requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[038] Throughout this specification, the term 'consisting of means consisting only of.

[039] A 'promoter' is defined as an array of nucleic acid control sequences that direct transcription of an operably linked nucleic acid. Promoters include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[040] The term 'operably linked' refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[041] 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 technology. 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 technology as it existed before the priority date of each claim of this

specification.

[042] Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the technology recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.

[043] In the context of the present specification the terms 'a' and 'an' are used to refer to one or more than one (ie, at least one) of the grammatical object of the article. By way of example, reference to 'an element' means one element, or more than one element.

[044] In the context of the present specification the term 'about' means that reference to a figure or value is not to be taken as an absolute figure or value, but includes margins of variation above or below the figure or value in line with what a skilled person would understand according to the art, including within typical margins of error or instrument limitation. In other words, use of the term 'about' is understood to refer to a range or approximation that a person or skilled in the art would consider to be equivalent to a recited value in the context of achieving the same function or result.

[045] Those skilled in the art will appreciate that the technology described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the technology includes all such variations and modifications. For the avoidance of doubt, the technology also includes all of the steps, features, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps, features and compounds.

[046] In order that the present technology may be more clearly understood, preferred embodiments will be described with reference to the following drawings and examples.

Brief Description of the Drawings

[047] Figure 1 : Plasmid map of pUC57 containing an expression cassette comprising the AOX1 promoter operably couple to a nucleic acid encoding Acaryochloris marina vBPO, a peroxisomal localisation tag and the DAS1 terminator sequence . [048] Figure 2: Gas chromatographs of a bromoform standard and extracts from cultures from a culture of P pastoris strains yJCIOO (sample 1), yJCIOO ACTA1 Gracilaria changii vBPO (sample 3), yJCIOO ACTA1 Chondrus crispus 615 vBPO (sample 6), and yJCIOO ACTA1 Alteromonas naphthalenivorans vBPO (sample 7).

[049] Figure 3: Gas chromatographs of a bromoform standard and extracts from cultures of P pastoris strains yJCIOO ACTA1 (sample 2), yJCIOO ACTA1 Corallina pilulifera vBPO (sample 4), yJCIOO ACTA1 Corallina officinalis vBPO (sample 5), yJCIOO ACTA1

Acaryochloris marina vBPO (sample 8), yJCIOO ACTA1 Laminaria digitata vBPO (sample 9), and yJCIOO ACTA1 Ascophyllum nodosum vBPO (sample 10).

[050] Figure 4: Gas chromatographs of extracts from a culture of P pastoris strains yJCIOO ACTA1 Acaryochloris marina vBPO (sample 8), yJCIOO ACTA1 Laminaria digitata vBPO (sample 9), and yJCIOO ACTA1 Ascophyllum nodosum vBPO (sample 10). yJCIOO ACTA1 Gracilaria changii vBPO (sample 3), yJCIOO ACTA1 Chondrus crispus 615 vBPO (sample 6), and yJCIOO ACTA1 Alteromonas naphthalenivorans vBPO (sample 7).

Description of Embodiments

[051] The biological production of bromoform using bromoperoxidse enzymes requires both one-carbon substrates such as methanol, formaldehyde, and formate, and hydrogen peroxide. Some yeast, such as Pichia pastoris, have the ability to grow on the one-carbon alcohol methanol and produce formaldehyde and formate as metabolic intermediates.

These native one-carbon metabolic processes are localised to the peroxisomes of Pichia pastoris to contain the toxicity of the intermediates.

[052] Bromoform is also highly toxic to cellular components and processes. Due to the availability of one-carbon substrates and hydrogen peroxide, and the need to minimise bromoform toxicity, the technology disclosed herein involves the localisation of

heterologously expressed vanadate-dependent bromoperoxidases to yeast peroxisomes, in one embodiment Pichia pastoris is used, using a peptide tag such as PEALIKSMTSKL.

[053] The toxicity associated with bromoform production is also limited by inducibly expressing the vanadate-dependent bromoperoxidases using methanol inducible promoters such as AOX1 , AOX2, CTA1 , DAS1 , DAS2, FLD, PMP20. Because these promoters are not active when yeast are grown with glucose as the carbon source, cells can accumulate to a high concentration free of bromoform-mediated toxicity, then switch to a bromoform production phase with growth on methanol. Methanol induces the expression of the vanadate-dependent bromoperoxidase and provides substrates to the enzyme in the form of one-carbon metabolites and hydrogen peroxide. [054] Accordingly, the technology relates to recombinant yeast capable of expressing at least one functional vanadate-dependent bromoperoxidase and which can then be used as a component of animal feed or an animal feed supplement. The feed and feed supplements can be used to reduce methane production in animals including ruminants such as cattle, sheep, goats, buffalo, deer, and camels.

Nucleic acids for expressing vanadate-dependent bromoperoxidase

[055] The practice of this technology involves the construction of recombinant nucleic acids and the expression of vBPO in yeast cells. Molecular cloning techniques to achieve this are known in the art. A wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids such as expression vectors are well-known to persons of skill. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found for example in Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994

Supplement).

[056] In order to express vBPO in a yeast, a nucleic acid encoding a vBPO is incorporated into an expression cassette. A typical expression cassette, which may be part of a larger nucleic acid construct such as an expression vector, contains a promoter operably linked to the desired vBPO encoding sequence and optionally other sequences such as a transcription terminator and a peroxisome targeting sequence.

[057] The promoter can be constitutive or inducible. Inducible promoters can be advantageous because the yeast cells can be grown to high densities before expression of the vBPO polypeptide is induced.

[058] A suitable constitutive promoter is the GAP(glyceraldehyde-3-phosphate

dehydrogenase)-promoter.

[059] Suitable inducible promoters are methanol inducible promoters. Methanol inducible promoters that can be used include the promoters from AOX1 (aldehyde oxidase 1), AOX2 (aldehyde oxidase 2), CTA1 (peroxisomal catalase), DAS1 (dihydroxyacetone synthase 1), DAS2 (dihydroxyacetone synthase 2), FLD (formaldehyde dehydrogenase), and PMP20 (peroxisome membrane protein which has glutathione peroxidase activity).

[060] In one embodiment the AOX1 promoter is used. The AOX1 promoter may comprise or consist of SEQ ID NO: 1 , or a sequence at least about 80%, at least 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 1. [061] Any known vBPO may be used, including nucleic acids encoding vBPO's from Acaryochloris marina (SEQ ID NO: 2), Alteromonas naphthalenivorans (SEQ ID NO: 3), Chondrus crispus 615 vBPO (SEQ ID NO: 4), Ascophyllum nodosum, Corallina officinalis vBPO (SEQ ID NO: 6) Corallina pilulifera (SEQ ID NO: 7), Gracilaria changii (SEQ ID NO:

8), or Laminaria digitate vBPO (SEQ ID NO: 9), Porphyra yezoensis (SEQ ID NO: 17), Chondrus crispus 963 (SEQ ID NO: 18) Synechococcus sp. CC9311 (SE ID NO: 19), Chondrus crispus 997 (SEQ ID NO: 20) or a sequence at least about 80%, at least 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% identical to any one of SEQ ID NOs: 2-9 or 17-20.

[062] In one embodiment the vBPO is G. changii vBPO (SEQ ID NO: 8) or a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to SEQ ID NO: 8.

[063] In one embodiment the vBPO is monomeric.

[064] In one embodiment the vBPO is A. marina vBPO (SEQ ID NO: 2) or a sequence at least about 80%, at least 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% identical to any one of SEQ ID NO: 2.

[065] Alternatively, nucleic acids encoding a vBPO can be prepared by any suitable method known in the art, including, for example, cloning and restriction of appropriate sequences or direct synthesis. In one embodiment, the desired nucleic acids encoding a vBPO are isolated by routine cloning methods. A nucleotide sequence encoding the vBPO enzyme (as provided below, for example) is used to construct probes that specifically hybridize to a bromoperoxidase gene in a genomic DNA sample, or to mRNA in a total RNA sample (e.g., in a Southern or Northern blot). Once the target nucleic acid is identified, it can be isolated according to standard methods known to those of skill in the art.

[066] The desired nucleic acids can also be cloned using amplification techniques known in the art. Examples of protocols sufficient to direct persons of skill through in vitro amplification methods, including the polymerase chain reaction (PCR) the ligase chain reaction (LCR).

[067] In some embodiments, it may be desirable to modify the bromoperoxidase nucleic acids of the invention. One of skill will recognize many ways of generating alterations in a given nucleic acid construct. Such well-known methods include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired

oligonucleotide (e.g., in conjunction with ligation and/or cloning to generate a nucleic acid encoding a modified vBPO). [068] In some embodiments the nucleic acids encoding the vBPO may be conservatively modified. With respect to nucleic acid sequences, conservatively modified refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are 'silent variations' which are one species of conservatively modified variations. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[069] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a 'conservatively modification' where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

[070] The following six groups each contain amino acids that are conservative

substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[071] A skilled person will recognise that other modifications can be made to the bromoperoxidase polypeptides or nucleic acids without diminishing their biological activity. Some modifications may be made to facilitate the cloning, expression, and the like. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids that form an epitope tag (e.g., poly His) placed on either terminus to facilitate purification or identification. In addition, one of skill will recognize that fusion proteins with various heterologous protein sequences can be prepared. For example, overexpression of a protein can lead to the accumulation of folding intermediates which have a tendency to aggregate. Production of fusion proteins including sequences, such as thioredoxin, can be used to facilitate proper folding.

[072] In some embodiments the expression cassette may include a nucleic acid sequence encoding a peroxisomal targeting signal. Typically the peroxisomal targeting signal is fused to the c-terminus of the vBPO. Accordingly the nucleic acid sequence encoding the peroxisomal targeting signal is in-frame with the sequence encoding the vBPO. However, in some embodiment a N-terminal peroxisome targeting sequence may be used.

[073] Any peroxisomal targeting sequence know in the art may be used, such as the following:

[074] In one embodiment the peroxisomal targeting sequence comprises or consists of PEALIKSMTSKL (SEQ ID NO:14).

[075] In some embodiments the expression cassette may lack a peroxisome targeting sequence. In these embodiments the vBPO is expressed and accumulates on the cytosol to access different carbon substrates for bromination. [076] The expression cassette may also include a transcription termination sequence that is functional in yeast. As exemplified herein the DAS1 terminator sequence (SEQ ID NO:

15) is useful.

[077] In some embodiments the expression cassette is suitable for integration into a yeast genome by methods known in the art. For example, the expression cassette can form part of a yeast integrative plasmid which can be introduced into the genome in linearized form. Alternatively PCR amplification of the expression cassette using extended primers with homology regions allowing integration into any locus. The expression cassette may be integrated into the genome using gene editing techniques such as CRISPR/Cas9 or using a meganuclease.

[078] In some embodiments the expression cassette is present in a plasmid vector.

Plasmid vectors for use in yeast are known in the art and suitable vectors include those containing a PARS1 origin of replications such as pUC57, pPpT4, plB2, pRS416, pRS415, pRS413, pRS406, pRS405, pRS403, pRS423, pRS426, pRS425, or variants of these.

[079] Suitable plasmids for use in yeast include integrating, replicating, centromere and episomal plasmids. Yeast Integrating plasmids lack an origin or replication (ORI) and are useful when the expression cassette is to be integrated directly into the host chromosome, for example via homologous recombination. Yeast Replicating plasmids contain an

Autonomously Replicating Sequence (ARS) derived from the yeast chromosome and replicate independently of the yeast chromosome. Yeast Centromere plasmids are considered low copy vectors and incorporate part of an ARS along with part of a centromere sequence. These vectors replicate as though they are small independent chromosomes and are thus typically found as a single copy but are stable without integration.

[080] In some embodiment the plasmid may contain the P. pastoris- specific autonomously replicating sequence (PARS1 , SEQ ID NO: 16). PARS1 permits transformation of P.

pastoris with higher efficiency than obtained following chromosomal integration by linearized DNA.

[081] Yeast episomal plasmids are high copy plasmids and contain a fragment from the 2 micron circle (a naturally occurring yeast plasmid) which allows 50 or more copies to stably propogate in each yeast cell. This is useful when high level expression of a polypeptide is required.

[082] The plasmid vector, or expression cassette can be transfected or electroporated into the yeast using methods known in the art such as the electroporation technique described in Lin-Cereghino, et al. BioTechniques 38, 44-48 (2005). Yeast strains

[083] It is envisaged that any yeast strain may be used. Suitable yeasts include Arxula adeninivorans (also known as Blastobotrys adeninivorans ), Candida boidinii, Ogataea polymorpha (also known as Hansenula polymorpha or Pichia angusta ), Kluyveromyces lactis, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica or Kluyveromyces marxianus.

[084] In some embodiments the yeast will be methylotrophic (able to grow on methanol). Suitable methylotrophic include Candida boidinii, Ogataea polymorpha, and Pichia pastoris.

[085] In one embodiment the metylotrophic yeast is Picihia pastoris.

[086] The yeast are modified to express at least one functional vanadate-dependent bromoperoxidase. In one embodiment the recombinant yeast comprises at least one copy of an expression cassette comprising a promoter operatively coupled to a nucleic acid sequence encoding a vanadate-dependent bromoperoxidase (vBPO) promoter is operatively coupled to the vBPO sequence in order to allow inducible expression of the vBPO.

[087] Hydrogen peroxide is produced as a by-product during growth on methanol, and is highly toxic to cells. Hydrogen peroxide is normally detoxified in yeast such as Pichia pastoris and Saccharomyces cerevisiae using a catalase enzyme such as the peroxisomal catalase CTA1. In some embodiments the yeast described herein have been modified by deletion of genes encoding a catalase enzyme such as CTA1. The effect of catalase deletion is that hydrogen peroxide accumulates and can be used as a substrate in bromoform production.

[088] In the absence of functional bromoperoxidase hydrogen peroxide accumulation reduces the growth rate and biomass yield of the yeast. Accordingly, deletion of CTA1 can be used to create a selective pressure or high-throughput screening method for expression of functional vanadate-dependent bromoperoxidases.

Bromoform production methods

[089] The conditions for culturing yeast both in the laboratory and on an industrial scale are well known to those in the art. Yeast are able to grow on many different carbon and energy sources such as glycerol, glucose and methanol which are commonly used in manufacturing processes. The choice of carbon-substrate and, therefore, the feasible operational range with respect to specific growth rate (m) and optimum productivity (qp) is dependent on the chosen promoter. Optimum conditions for the production of a

recombinant protein in P. pastoris differ according to the target molecule and promoter [090] Strains expressing a heterologous gene under the control of the AOX1 -promoter utilise methanol, which is required both as an inducer, and for biomass growth and production

[091] In embodiments where a methanol induced promoter is used methanol acts as an inducer for vBPO expression and, at the same time, as a carbon and energy source. Thus, induction and vBPO production of heterologous protein are interconnected with substrate utilisation and biomass growth. Consequently, vBPO expression can be controlled by modulating the level of methanol on the culture medium as this will tend to reduce as the yeast grows.

[092] Bromoform can be produced in yeast transformed with one or more of the nucleic acids described above. The production of bromoform involves culturing the yeast in the absence of methanol, adding methanol to the culture to induce the expression of the vBPO and then further culturing the yeast to allow the expressed vBPO to produce bromoform.

[093] In other embodiments cultivating the yeast for bromoform production may follow a more involved strategy that requires an initial batch phase for biomass growth with, for example glycerol or glucose. This can be followed by an optional fedbatch phase for further biomass enhancement with for example glycerol or glucose. In order to instigate vBPO production an optional methanol-induced adaptation (transition) phase may be used to transition the yeast from for example glycerol or glucose to methanol. This is followed by a production phase in fedbatch mode during which the methanol level is monitored and additional methanol is added. Generally, a high initial biomass and a low specific growth rate during production are favourable for vBPO expression and maximum bromoform production.

[094] In order for bromoform to be produced the culture medium contains methanol, both as a substrate and to induce vBPO expression in embodiments where a methanol inducible promoter is used. The methanol concentration can be from 0.5% (v/v) to about 5% (v/v), for example the methanol concentration may be about 0.5% (v/v), 1 % (v/v), 1.5% (v/v), 2% (v/v), 2.5% (v/v), 3% (v/v), 3.5% (v/v), 4% (v/v), 4.5% (v/v), or about 5% (v/v). The media also includes a source of vanadium, such as sodium orthovanadate. Typically the concentration of sodium orthovanadate is about 0.5mM to 5.0mM, for example about 0.5 mM, 1 mM, 1.5 mM, 2 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or about 5mmM. Further sodium or potassium bromide (or a mixture thereof) is added to the culture medium at a

concentration from about 1 mm to about 500mM, for example 1 mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM, 20 mM, 30 mM, 40mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM or about500 mM. [095] In some embodiments the bromoform concentration in the yeast culture is about 1 mM to at least about 400 mM. For example the maximum bromoform concentration in the yeast culture may be about 1 mM, 100 mM, 250 mM, 500 mM, 750 mM, 1 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, or at least about 400 mM.

[096] In some embodiments the methods may involve harvesting of the bromoform containing yeast from the culture. Harvesting can be by any method known in the art such as centrifugation, for example in a centrifugal pump (a separator), filtration, or settling.

[097] The harvested yeast may be further processed for example by evaporation, air drying, freeze drying, or oven drying. In some embodiments the yeast may be processed into a yeast extract, for example by lysing the yeast (for example by heat, mechanical means or autolysis) and extracting their cell contents, this typically also involves removal of cell walls.

Extracts, Supplements, and Animal Feeds

[098] The harvested yeast, whether subjected to further processing or not, are useful as an animal feed supplement due to the bromoform content which inhibits the production of methane by microbes present in the digestive system of the animal.

[099] An animal feed supplement is a concentrated additive or premix which is either added to animal feed (for example in a feedlot) or is available to an animal (for example a lick-block). Typically, the animal feed supplement containing the yeast or yeast extract is in the form of a powder or compacted or granulated solid. In practice, the supplement may be added to the ration, e.g. as a top-dress, or it may be used in the preparation or manufacture of products such as compounded animal feeds or lick blocks.

[0100] In some embodiments the yeast may be added to an animal's drinking water.

[0101] In one embodiment the harvested yeast may be used as a supplement and fed directly to cattle. In other embodiments the harvested yeast can be added to known supplements such as lick blocks, grain, molasses, silage, cotton seed meal, lupins and the like. Supplements containing the harvested yeast (or extracts thereof) are useful in reducing methane production in grazing animals.

[0102] The harvested yeast may also be used as a component of animal feed, for example animal feed suitable for use in a feedlot. Methods of reducing methane production

[0103] There is also provided a method for reducing methane production in an animal. The method comprises administering to the animal an effective amount of the harvested yeast. This can occur by feeding the yeast to the animal directly or as part of an animal feed supplement, or animal feed.

[0104] An 'effective amount' is a quantity of the harvested yeast or extract thereof which is sufficient to reduce methane production by an animal in comparison with a control.

[0105] In some embodiments the concentration of bromoform required to inhibit

methanogenesis in the rumen is about 0.5 mM - 10 mM, for example 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM. A skilled person can calculate the amount of yeast to be fed to each animal from the bromoform concentration in the yeast, the amount of yeast added to the feed and the approximate rumen volume.

[0106] In one embodiment, the yeast or yeast extract is administered at a dose of about 0.05% to about 20% or the organic matter fed to the animal. For example, the dose may be about 0.05%, 0.1%, 0.5%., 1%, 2%, 3%, 4%, or 5% of the organic matter fed to the animal.

[0107] Advantageously, animals prefer the taste of yeast and will preferentially eat feed supplemented with yeast over feed supplemented with seaweed.

[0108] In another embodiment the yeast or yeast extract is administered in a dose of about 0.1 - 10 g/kg body weight per day, for example 0.1 , 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 g/kg body weight per day.

[0109] The present methods may comprise administration of the yeast or yeast extract for 1 , 5, 10, 25, 50, 100, 125, 150, 200, 250, 270, 300, 325, 350 days or more.

[0110] The effective amount of the yeast or yeast extract can be administered to said ruminant animal in one or more doses, for example one or more doses on a daily basis.

[0111] The yeast or yeast extract can be administered daily, every other day, every other two days, etc., without departing from the scope of the invention. In some embodiments the yeast or yeast extract, either alone or as part of an animal supplement of animal feed is available to the animals to consume ad libitum.

Animals

[0112] The methods are particularly useful for reducing methane production in ruminant animals such as ruminants such as bovines (e.g. cattle and buffalo), ovines (e.g. sheep) caprine (e.g. a goat), equine (e.g. a horse or donkey) , cervids (e.g. deer), and camelids (e.g. camels, llamas, alpacas). In some embodiments the methods can be used to reduce methane formation in non-ruminant animals such as porcine and avian animals including poultry.

[0113] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Examples

Example 1 : Bromoperoxidase expression vectors

[0114] Bromoperoxidase expression vectors were synthesised by on the pUC57 backbone, with individual bromoperoxidase genes cloned between the AOX1 promoter and DAS1 terminator sequences using homology-mediated recombination. Vector configurations are illustrated in Figure 1 illustrating the position of the Acaryochloris marina brom peroxidase coding sequence.

[0115] A series of vectors were synthesised in the same way but for the identity of the bromoperoxidase gene. Vectors were synthesised to express each of the following bromoperoxidases: Acaryochloris marina (SEQ ID NO: 2), Alteromonas naphthalenivorans (SEQ ID NO: 3), Chondrus crispus 615 (SEQ ID NO: 4), Ascophyllum nodosum (SEQ ID NO: 5), Corallina (SEQ ID NO: 6), Corallina pilulifera (SEQ ID NO: 7), Gracilaria changii (SEQ ID NO: 8), Laminaria digitate (SEQ ID NO: 9).

[0116] The plasmids were synthesised to express the bromoperoxidase with a C-terminal PEALIKSMTSKL localisation peptide (SEQ ID NO: 14). The expression vectors include the coding sequence for the localisation peptide in frame with the coding sequence of the bromoperoxidase so that he expressed vBPO is targeted to the peroxisome.

[0117] The coding sequence for the brompoeroxidase is operativey coupled with the Pichia pastoris AOX1 promoter sequence to enable methanol inducible expression of the bromoperoxidase.

[0118] The AOX1 promoter sequence used in this example is SEQ ID NO: 1.

Transcriptional termination was mediated by the DAS1 terminator sequence (SEQ ID NO: 15). Plasmid replication was mediated by the PARS1 sequence (SEQ ID NO: 16).

[0119] A zeocin resistance gene was included on the plasmid to enable selection. Example 2 Bromoperoxidase expression

[0120] Pichia pastoris yJCIOO (wild-type) cells with a disrupted CTA1 gene were transformed with one of the vectors from Example 1 using electroporation using the technique of Lin et al (BioTechniques, 2005, vol 38, pages 44-48. The transformed cells were selected on yeast extract (1 % w/v), peptone (2 % w/v), dextrose (2 % w/v) agar (2 % w/v) plates with 200 pg/mL zeocin.

[0121] Colonies transformed with each of the eight bromoperoxidase expression vectors were then grown in liquid medium alongside empty vector control strains for the collection of samples for bromoform identification and quantification. Cells were pre-cultured twice on liquid minimal medium with 1x Yeast Nitrogen Base (YNB, Sigma Y0626), 1 % w/v glucose, and 100 pg/mL zeocin. Each strain was then inoculated at an optical density of 0.2 at 600 nm (OD600) in 250 mL baffled shake-flasks with 20 mL of liquid medium containing 1 % w/v glucose, 1 % v/v methanol, 1x YNB, 50 pg/mL zeocin, 1 mM sodium orthovanadate, and 200 mM sodium bromide. Flasks were shaken at 200 rpm, 30 °C in a 25 mm orbital Infors HT shaking incubator.

[0122] After 48 hours, the OD600 of each culture was measured and 1 mL samples were collected by centrifugation at 14,000 x g for 2 minutes with supernatants transferred to fresh microtubes. Samples were stored at - 20 °C prior to analysis.

[0123] Bromoform presence/absence and relative abundance was assayed using gas chromatography mass spectrometry as previously described (Machado et al. J. Appl.

Phycol. 28, 3117-3126 (2016); Paul et al, Mar. Ecol. Prog. Ser. 306, 87-101 (2006)). The relative peak sizes of bromoform from each of the samples are shown in Figures 2-4.

Sample numbers correlate to Pichia pastoris yeast strains as follows:

1. yJCIOO,

2. yJCIOO ACTA1 ,

3. yJCIOO ACTA1 Gracilaria changii vBPO,

4. yJC 100 ACTA 1 Corallina pilulifera vBPO,

5. yJC100 ACTA1 Corallina officinalis vBPO,

6. yJCIOO ACTA1 Chondrus crispus 615 vBPO,

7. yJCIOO ACTA1 Alteromonas naphthalenivorans vBPO,

8. yJCIOO ACTA1 Acaryochloris marina vBPO,

9. yJCIOO ACTA1 Laminaria digitata vBPO, 10. yJCIOO DOTA1 Ascophyllum nodosum vBPO.

[0124] The GCMS elution profiles in Figure 4 indicate that sample 8 (vBPO from A. marina) had the largest bromoform peak, which corresponds to 4 mM when pure external bromoform standards were used to make a calibration curve. Injection of supernatants from the two control strains or a water blank sample did not result in peaks corresponding to the bromoform standard. Bromoform peaks were also identified in some of the other samples (such as 9 and 10), but these were at least half as large as the sample - 8 peak. The final OD600 readings from each strain were not significantly different.

[0125] In contrast to the protein structures of the other vBPOs that were tested, the A. marina vBPO is monomeric.