Transgenic insects and preparations thereof

Abstract

There is provided a transgenic insect comprising a heterologous nucleic acid encoding a fungal xylanase, wherein the heterologous nucleic acid is integrated in the insect genome, wherein the heterologous nucleic acid is operably linked to a promoter, and expression of the xylanase allows for the production of an active fungal xylanase in the insect. The insect or xylanase preparation made from the insect is used for animal feed or feed additives and other industrial applications.

Description

TRANSGENIC INSECTS AND PREPARATIONS THEREOFCross reference to related application

[0001] This application claims the benefit of Australian Provisional Application No. 2023903731 filed 20 November 2023, the entire contents of which is incorporated by reference herein.Technical Field

[0002] The technology relates to transgenic insects, for example black soldier fly larvae (Hermetia illucens), expressing fungal xylanase and use of the transgenic insects or xylanase preparation made from the insects for animal feed or feed additives and other industrial applications.Background

[0003] Xylan which consists of a backbone of - ,4-linked xylose units is a major component of hemicellulose and plant fiber. The processing of plant derived non-starch polysaccharides (NSP) found in cereal grains (e.g., wheat, barley or corn) require carbohydrase enzymes that depolymerize xylan. Xylanases (endo-1 ,4-p-D-xylanase) hydrolyse glycosidic linkages on xylan and convert it into smaller oligosaccharides that are easier to process. These xylanases are commonly used for a variety of purposes such as feed supplements to help animals break down plant material, for bleaching paper pulp, for extraction and clarification of juices, and for ethanol production.

[0004] Xylanase enzymes have been added to animal feed to increase the efficiency of digestion and assimilation of nutrients. During digestion of feed grains (e.g., wheat and barley) NSP including xylan increases the viscosity of the digesta in the absence of added exogenous enzymes. This interferes with the diffusion of the digestive enzymes to the feed and the subsequent assimilation of the nutrients. The highly viscous digesta increases the occurrence of sticky stool, which increases the likelihood of disease and causes effluent run-off problems. The addition of xylanase in animal feed breaks down the xylan and decreases the viscosity of the digesta, thereby helping to alleviate these issues.

[0005] Current production methods for xylanase are primarily microbial with fungi and bacteria commonly used to produce xylanases via either submerged fermentation (the predominant method) or solid-state fermentation. Submerged fermentation involves culturing organisms in bioreactors that are filled with liquid nutrients. This system is highlycontrolled, however, due to the need for specialized bioreactors, and the technique requires sophisticated and expensive infrastructure for scaling up production.

[0006] Accordingly, there is a need for methods of producing xylanase in an efficient and cost-effective manner with comparably small infrastructure requirements.

[0007] The present inventors have developed methods of producing xylanase in transgenic insects. The methods generate more value from organic waste because insects, such as black soldier flies, are able to survive and proliferate on a diet of organic waste and several insect species can be directly used as suitable livestock feed or feed ingredients. The methods significantly reduce resource requirements and the time required for producing xylanase for feed additive and the xylanase preparation made from insects can be used for other industrial processes.Summary

[0008] The invention generally relates to a method of producing a functional xylanase in transgenic insects. The invention also relates to a method of improving nutrient availability or digestibility of an animal feed by combining an effective amount of xylanase prepared from the transgenic insects with the animal feed.

[0009] In a first aspect, the invention relates to a transgenic insect capable of expressing an active fungal xylanase, the insect comprising a heterologous nucleic acid encoding the fungal xylanase, wherein the heterologous nucleic acid is integrated in the insect genome, and wherein the heterologous nucleic acid is operably linked to a promoter.

[0010] The fungal xylanase may be from Trichoderma reesei.

[0011] In one embodiment, the heterologous nucleic acid comprises SEQ ID NO: 1 or a sequence at least 80%, 85%, 90%, 95%, 97%, or 99% identical to SEQ ID NO: 1 .

[0012] In one embodiment, the heterologous nucleic acid comprises SEQ ID NO: 2 or a sequence at least 80%, 85%, 90%, 95%, 97%, or 99% identical to SEQ ID NO: 2.

[0013] The heterologous nucleic acid may further comprise a sequence encoding a signal peptide in frame with the sequence encoding the fungal xylanase. The signal peptide may be the larval cuticle protein 9 signal peptide or a sequence at least 80%, 85%, 90%, 95%, 97%, or 99% identical to larval cuticle protein 9.

[0014] The promoter may be a short tubulin alpha promoter.

[0015] The insect may be selected from the genus Hermetia, Drosophila, or Tenebrio. For example, the insect may be Hermetia illucens, Drosophila melanogaster or Tenebrio molitor.

[0016] In a second aspect, the invention relates to a xylanase preparation comprising the transgenic insect of the first aspect, or a portion thereof.

[0017] The transgenic insect of the xylanase preparation may be fully or partially dried. The transgenic insect of the xylanase preparation may be in the form of a meal or a powder. The xylanase preparation may further comprise one or more of a preservative, anti-caking agent or surfactant.

[0018] In a third aspect, the invention relates to a feed additive composition comprising a xylanase preparation of the second aspect.

[0019] In a fourth aspect, the invention relates to a method for improving nutrient availability or digestibility of an animal feed comprising combining an effective amount of a xylanase preparation of the second aspect or a feed additive composition of the third aspect with the animal feed. The method further comprises combining at least one additional ingredient selected from vitamins and minerals.

[0020] The animal may be, for example, chicken, pig, cattle, sheep, goat, shellfish and fish.Definitions

[0021] 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.

[0022] Throughout this specification, the term 'consisting essentially of' means the inclusion of the stated element(s), integer(s), or step(s), but other element(s), integer(s), or step(s) that do not materially alter or contribute to the working of the invention may also be included.

[0023] Throughout this specification, the term 'consisting of' means consisting only of.

[0024] Any discussion of documents, acts, materials, devices, articles, or the like included in the present specification is solely to provide 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.

[0025] 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.

[0026] In the context of the present specification, the terms 'a' and 'an' refer to one or more than one (i.e., at least one) of the grammatical object of the article. For example, a reference to 'an element' means one element or more than one element.

[0027] 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, the 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.

[0028] 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.

[0029] 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.

[0030] The terms 'increase' or 'improve' (and grammatical variations thereof) as used herein describe an increase or improvement in a specified parameter by feeding to an animal a xylanase preparation or feed additive composition as described herein, wherein the specified parameter is elevated as compared with an animal not fed xylanase or a xylanase preparation or feed additive composition as described herein, for example, is fed a conventional feed that does not comprise exogenous xylanase.

[0031] 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 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 the said steps, features, and compounds.

[0032] 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

[0033] Figure 1 : Initial xylanase enzyme activity of two Trichoderma reesei xylanases. Strains of the Xylan2-1 and Xylan2-2 flies were compared and found to have no significant difference (p = 0.9742). All samples were left to incubate for 60 minutes for the reaction to occur before Tris stopping buffer was added. Xylan 2-1 = transgenic xylanase flies with construct containing a propeptide region; Xylan 2-2 = transgenic xylanase flies without a propeptide region; EV = empty vector flies; Pos = positive control provided by the XylX6 assay kit; RB = reagent blank. Samples with shifts in absorbance less than 0.15 are considered instrument noise. Xylanase activity was determined via commercially available xylanase assay kit (XylX6 method) from Megazyme and determined via microplate reader (Spectrostar nano, BMG Labtech) at 400nm.

[0034] Figure 2: Trichoderma reesei xylanase activity over time (0, 25, 50, 75, 100, and 125 minutes). Xylan 2-1 = transgenic xylanase flies with construct containing a propeptide region; Xylan 2-2 = transgenic xylanase flies without a propeptide region; EV = empty vector flies; Pos = positive control provided by the XylX6 assay kit; RB = reagent blank. Samples with shifts in absorbance less than 0.15 are considered instrument noise. Xylanase activity was determined via commercially available xylanase assay kit from Megazyme and determined via microplate reader (Spectrostar nano, BMG Labtech) at 400nm.Description of Embodiments

[0035] The technology described herein generally relates to transgenic insects expressing a xylanase enzyme. The insect, preferably in the larval or pupal stage, is used as the base for a xylanase preparation containing the expressed xylanase. The xylanase preparation can be used as a feed additive for livestock animals to improve nutrient availability or digestibility of an animal feed.

[0036] Described herein are insects (e.g., Hermetia illucens) that are genetically engineered to express a functional xylanase from fungi e.g., Trichoderma reesei). Transgenic insects expressing a functional xylanase are capable of hydrolysing glycosidic linkages on xylan and convert it into smaller oligosaccharides that are easier to process. The xylanase preparation described herein when fed as or combined with an animal feed improves digestibility and / or nutrient availability of the animal feed and reduces digesta viscosity by breaking down non-starch polysaccharides (NSP) found in cereal grains (e.g.,wheat, barley or corn). The use of transgenic insects expressing xylanase as animal feed or additive can increase the efficiency of digestion and assimilation of nutrients.Xylanase

[0037] In the context of this disclosure, xylanase (endo-1 ,4-p-D-xylanase) refers to the enzyme classification E.C.3.2.1 .8. The composition of a plant cell wall is complex and variable. Polysaccharides are mainly found in the form of long chains of cellulose which is the main structural component of the plant cell wall, hemicellulose which comprises various P-xylan chains, and pectin. Xylanase has the ability to catalyze cleavage of xylan at one or more of various positions of xylan's carbohydrate backbone, including branched xylans and xylooligosaccharides.

[0038] Xylanase activity may be determined by any suitable assay. Generally, assay-pH and assay-temperature may be adapted to the enzyme. Xylanase may be of bacterial or fungal origin. It is contemplated that any xylanase known in the art may be used in the recombinant insects described herein and a skilled person will be able to identify suitable xylanase. In a preferred embodiment, the xylanase is thermostable. In another preferred embodiment, the xylanase is pH stable (e.g., an acid pH). Even more preferably, the xylanase exhibits the property of increased thermostability (e.g., the enzymes can tolerate high temperature during food pelleting process for sterilization) while maintaining pH and temperature optima that are typically found under physiological conditions (e.g., in the digestive system of an animal).

[0039] Microbial xylanase enzymes may be derived from bacteria or fungi. Suitable fungal xylanase enzymes include but is not limited to filamentous fungi of the genera Aspergillus, Disporotrichum, Penicillium, Neurospora, Fusarium and Trichoderma. Preferably, the fungal xylanase derives from Aspergillus niger. Aspergillus awamori, Aspergillus aculeatus. Aspergillus tubigensis, Disporotrichum dimorphosporu, Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma harzianum, Trichoderma lignorum, and Trichoderma viride. More preferably, the xylanase is from Trichoderma reesei. Suitable bacterial xylanase enzymes include but is not limited to the genus Bacillus, for example, Bacillus amyloliquefaciens, Bacillus circulans, Bacillus halmapalus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, Bacillus natto, Bacillus mesentericus, Bacillus coagulans, Bacillus pumilus, and Bacillus firmus.

[0040] In one embodiment preferably the xylanase is from Trichoderma reesei.

[0041] Fungal xylanases have a pH optimum in the range of between pH 3.5 - 5.5 as compared to bacterial xylanases which generally have a pH optimum in the range of pH 5.0 - 7.0. Fungal xylanases generally have a broader pH stability range (pH 3 - 10) compared to bacterial xylanases (pH 5.0 - 7.5). Fungal xylanases generally have a temperature optimum of about 50°C. Bacterial xylanases generally have a temperature optimum between 50°C and 70°C.

[0042] The xylanase enzymes as described herein may contain various sequence changes from the wild-type or naturally occurring xylanases. In this context, the term "% identity" refers to the level of nucleic acid or amino acid sequence identity between the modified xylanase and the wild-type xylanase. For example, modified xylanase may have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to its wild-type counterpart. In some embodiments, the modifications do not alter the enzymatic activity, specificity, thermostability, or pH stability of the xylanase.

[0043] In one embodiment, the xylanase is a Trichoderma reesei xylanase, for example a Trichoderma reesei xylanase encoded by SEQ ID NO: 1 (containing a native propeptide region) or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 1 . In another embodiment, the xylanase is a Trichoderma reesei xylanase encoded by SEQ ID NO: 2 (without a native propeptide region) or a sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 2.

[0044] In some embodiments, it may be desirable to modify the xylanase. One of skill in the art will recognize many ways of generating alterations in a given nucleic acid construct. Such well-known methods include site-directed mutagenesis, FOR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of the desired oligonucleotide (e.g., in conjunction with ligation and / or cloning to generate a nucleic acid encoding a modified xylanase), and codon optimisation for generating high expression constructs.

[0045] In some embodiments, the nucleic acids encoding the xylanase 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 everyposition 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 can be modified to yield a functionally identical molecule (except AUG, which is ordinarily the only codon for methionine). Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0046] 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 'conservative 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.

[0047] 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); and6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0048] A skilled person will recognise that other modifications can be made to the xylanase 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.

[0049] In some embodiments, the xylanase is not a thermostable xylanase. In some embodiments, the xylanase is a thermostable xylanase. In some embodiments, the xylanase can tolerate a wide pH range (e.g., is active across a wide pH range, including acidic pH).

[0050] Manipulating the properties of the xylanase enzymes described herein can be achieved using modern protein engineering tools known in the art. Properties such as optimum pH, thermotolerance, and operational stability may be targeted to modify xylanases.

[0051] In order to express xylanase in an insect, a nucleic acid encoding the xylanase is incorporated into an expression cassette or expression vector. 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 a nucleic acid encoding the xylanase and optionally other sequences such as a transcription terminator and a sequence encoding a signal peptide.

[0052] The promoter can be constitutive or inducible. Inducible promoters can be advantageous because the insect cells can be grown to high densities before the xylanase polypeptide expression is induced.

[0053] Examples of suitable constitutive promoters are actin 5C promoter, 5'LTR of a COPIA element, or the short tubulin alpha promoter.

[0054] In one embodiment, the short tubulin alpha promoter is used.

[0055] As the shortened alpha tubulin promoter is effective with low potential toxicity, it is apparent that other ubiquitous promoters which have low potential toxicity and that drive the overexpression of enzymes in all tissues can also be used.

[0056] Suitable inducible promoters include metallothionen, HSp-70 promoter, and tetracycline operator promoters.

[0057] In some embodiments, the xylanase is secreted. Xylanase for secretion may fuse, either directly or via a linker sequence, to a signal peptide, for example, at the N-terminus that directs the protein into the endoplasmic reticulum, which is the first step in the pathway leading to secretion from the cell. The signal peptide may be fused to the xylanase, or the nucleic acid sequence encoding the xylanase signal sequence may be replaced by a nucleic acid sequence encoding a heterologous signal peptide.

[0058] Numerous signal peptides are known in the art and include signal peptides from multiple sources such as signal peptides from human beta-interferon, human placentalalkaline phosphatase, D. melanogaster cuticle protein II, or larval cuticle protein 9, Bombyxin. mori bombyxin, Heliothis virescens juvenile hormone esterase, Trichoplusia ni acidic juvenile hormone-suppressible hemolymph protein, flesh fly sarcotoxin IA, honeybee melittin, Manduca sexta adipokinetic hormone, Lucilia cuprina (Australian sheep blowfly) chymotrypsin, AcMNPV GP67, AcMNPV EGT, Spodoptera litura NPV EFP (envelope fusion protein), and the venom neurotoxin signal peptides from the mite toxin encoded by tox21 A and neurotoxins AalT, LqhlT2, and Bj IT (from the scorpion Hottentota judaicus). Signal peptides from different sources can be compared to identify a sequence that drives optimal xylanase secretion levels. For example, the native TXP-1 signal peptide and the signal peptides of D. melanogaster cuticle protein II and the tox21 A may be combined.

[0059] In one embodiment, the signal peptide is from larval cuticle protein 9.

[0060] In other embodiments, the xylanase is expressed intracellularly and in these embodiments the xylanase in not fused to a signal peptide.Insects

[0061] One advantage of the present invention is that xylanase is expressed in insect species that can be easily cultivated on food or agricultural waste. Accordingly, any insect used for “bioconversion” of waste into insect biomass can be used to express xylanase. Presently, only a few insect species are commercially used for insect-based bioconversion of food waste, with black soldier fly larvae (Hermetia illucens) being the most commonly used species. However, it is envisaged that any insect species amendable to genetic modification can be used to express xylanase and form the basis of a xylanase preparation. Considering the diversity of food and agricultural waste it is envisaged that waste-to-insect pairings to maximize both bioconversion and insect biomass (and hence xylanase) production may be made by the skilled person.

[0062] For example, vegetative food wastes can be fed to both black soldier fly larvae and mealworm larvae, but this waste is too low in protein content for housefly larvae. Conversely, restaurant and kitchen wastes containing meat are well suited for housefly and black soldier fly larvae but are too wet for mealworms, which can get moisture directly from the air and thus perform optimally in drier wastes. Further, black soldier fly larvae are tolerant of wet wastes and high temperatures (from bacterial and colony metabolism), allowing them to capitalize on many waste streams. In one embodiment, the insect species is cultivated on food or agricultural waste rich in xylans (e.g., cereal grains such as wheat, rye, barley, oat, rice, corn etc), to increase growth.

[0063] The choice of insect is based on a variety of factors including one or more of fast growth; high reproduction rates; high fecundity; large biomass; gregarious nature; short life cycle; disease resistance; ease of harvest; tolerate high stocking densities; have high food conversion rates; do not require excessive heating for reproduction and growth; and consume diets that are readily available and inexpensive to produce. In some embodiments, the insects provide maximum dry yield. These characteristics ensure that the selected insects will rapidly increase in volume by both quantity and size. Further, the insect's rapid rate of growth and reproduction will contribute to the size of the harvest as well as provide replacement stock for a new crop of insects.

[0064] The insects may be cockroaches, flies, beetles, worms, larval stages of other flying insects such as mealworms, caterpillars, etc. The most desirable insects to produce xylanase in terms of composition, size, reproduction, palatability, and lack of known toxins are typically species found within the orders Blattodea (cockroaches), Orthoptera (grasshoppers, locusts, katydids, crickets), Diptera (flies), and Lepidoptera (moths and butterflies).

[0065] It is envisaged that any dipteran insect may be used to express the xylanase. Suitable dipteran insects include soldier flies, robber flies, bee flies, hover flies, fruit flies, vinegar flies, and blowflies.

[0066] In one embodiment, the insect is a fruit fly Drosophila sp.), black soldier fly (Hermetia sp, Hermetia illucens), or house fly. Preferably the insect is a black soldier fly.

[0067] In other embodiments, the insect may be a mealworm, for example, of the genus Tenebrio. In one embodiment, the mealworm is Tenebrio molitor.

[0068] The xylanase can be expressed in one or any combination of the eggs, larvae, pupae, and adults. In one embodiment, the insect expresses the xylanase to improve digestibility of xylanase present in the insect's own diet for improved feed conversion ratios.Transformation of Insects

[0069] In general, transformation is a process in which exogenous DNA sequences are introduced into the insect germ line. Any xylanase nucleic acid that can be integrated into the insect germ line can be utilized in accordance with the present invention. Numerous methods for transforming insects and nucleic acid vectors that can be used for insect transformation are known in the art.

[0070] To produce heritable changes, the xylanase or expression vector containing the xylanase must be transformed into the insect during early development, prior to germ cellformation, or directly within the germ line precursors. In flies, this can be achieved through three experimental approaches. First, editing tools are physically introduced via embryo transformation by microinjection, requiring outcrossing to ensure germ line transmission and identify unique events. Alternatively, in-vivo remobilization allows gene targeting of complicated gene targeting constructs. Finally, in-vivo upgrading allows novel material to be incorporated into previously established “docking” sites. The latter two are performed through genetic crosses, conveniently circumventing any physical manipulation.

[0071] The first step for any kind of genome engineering introduced into insects typically involves embryo microinjection. Fertilized embryos are injected at a very specific developmental stage, the multinucleated syncytial one cell stage, just before cellularization. This timing maximizes the number of germ cells to be transformed before cellular membranes omit accessibility to the injected material. Injected insects must be outcrossed to identify germ line transmission of the engineered changes to the next generation. This strategy has been successful for many germ line-based manipulations in insects.

[0072] Microinjection can be performed as "co-injection” or “direct” injections. During “coinjection,” two components are introduced: the xylanase nucleic acid, expression cassette of an expression vector, and a catalyst, i.e., recombinase, integrase, or nuclease with or without guide RNA. The catalyst can be provided in trans, as plasmid DNA encoding a promoter driving the catalyst, e.g., <t>C31 integrase, Cre recombinase, Flp recombinase, TALEN, or Cas9. Such plasmids are commonly known as “helper” plasmids, alternatively, the catalyst may be encoded by a nucleic acid that forms part of the expression cassette or expression vector. In other embodiments, mRNA encoding the catalyst can be injected, i.e., <t>C31 integrase, Bxb1 integrase, ZFN, TALEN, or RGN1. Finally, the purified protein could be injected, e.g., Cas9. Co-injections limit catalyst activity over time (i.e., through dilution), which is often advantageous.

[0073] Alternative to co-injections, expression of the catalyst from a genomic source, i.e., “direct” injections, can be accomplished by injecting a genome engineering template without a helper plasmid; this requires that the catalyst (enzyme coding sequence) must have been established in the genome first before any genome engineering experiment can be performed. "Direct” injections can utilise <t>C31 integrase, ZFN, and Cas9. While this approach simplifies the procedure, the catalyst-bearing chromosome should be removed by genetic outcrossing.

[0074] In some embodiments, transformation is performed using P-elements. A P-element is a transposon that is present in D. melanogaster and is used widely for mutagenesis and the creation of genetically modified flies. A P-element is a class II transposon, which meansthat its movement within the genome is made possible by a transposase. The complete element is 2907 bp and is autonomous because it encodes a functional transposase. Non- autonomous P-elements which lack a functional transposase gene due to mutation also exist. Non-autonomous P-elements can still move within the genome if there are autonomous elements to produce transposase. A P-element can be identified by the presence of terminal 31 -bp inverted repeats and the 8 bp direct repeats in movement into and out of DNA sequence produces.

[0075] Naturally-occurring P-elements typically contain a coding sequence for the enzyme transposase and recognition sequences for transposase action. Transposase is an enzyme that regulates and catalyzes the excision of a P-element from the host DNA, cutting at two recognition sites and then reinserts randomly. In general, to use P-elements as useful and controllable genetic tools, the two parts of a P-element are separated to prevent uncontrolled transposition. The normal genetic tools are, therefore, DNA coding for transposase with no transposase recognition sequences so it cannot insert, and a P- element construct. P-element constructs typically comprise a reporter useful for selecting transformants (e.g., white+, yellow+, etc.) and transposase recognition sequences. P- element constructs may further comprise a gene of interest, a bacterial reporter gene (e.g., a gene encoding for antibiotic resistance), an origin of replication, etc.

[0076] In some embodiments, transformation is performed using piggyBac elements. A piggyBac element is a short inverted terminal repeat (ITR) transposable element that is approximately 2.5 kb long and comprises short (e.g., 13-bp) ITR sequences and an ORF. It is part of a subclass of ITR elements that insert exclusively into TTAA target sites. On insertion, the target site is duplicated with excision occurring in a precise fashion, restoring the insertion site. Beyond this functional similarity, the TTAA elements share no apparent structural identities. piggyBac vectors have been shown to mediate germ line transformation in insect species.

[0077] A system involving Cre and FLP that allows for the study of two genes at identical places in the genome can also be used. In this system, an insect line is created by P- element insertion that contains the two transgenes of interest flanked by either loxP or FRT sequences. Under Cre expression, one transgene is removed, while under FLP expression, the other transgene is removed. Each remaining transgene is then left in the same chromosomal context.

[0078] In some embodiments, an approach to the site-specific integration problem is the use of homologous recombination. In general, the frequency of homologous recombination has been too low to be of practical use in insects. However, in some embodiments, thefrequency of homologous recombination can be boosted using P-element transformation to insert a construct containing the gene to be targeted, engineered with an l-Scel cutting site, and flanked by two FRT sites. This construct can then be mobilized as a circular DNA molecule by expression of FLP and made linear by the expression of l-Scel, increasing the targeted recombination frequency. In this system, a separate P-element insertion carrying the homologous DNA engineered with l-Scel and FLP sites is required for each gene to be targeted. By this method, a targeted event could be obtained at a frequency of about 1 in 500-30,000 gametes from the female germ line.

[0079] In some embodiments, the FLP / FRT system has been used to insert genes into any desired place in the genome. An integration frequency of up to 5% into a FRT site in the genome can be obtained when the target DNA is mobilized from elsewhere in the genome by FLP excision.

[0080] In some embodiments, transformation is performed using integrase-mediated systems. The site-specific integrase from phage cpC31 has been shown to function at high frequency, requires no cofactors, and mediates recombination between two sequences, the attB, and attP sites, to create stable recombinants. Both intra- and inter-molecular recombination occur at high frequencies, and essentially no reversion of the reaction occurs. It has been demonstrated that the integrase can recognize and integrate into endogenous pseudo attP sites that have partial identity to attP.

[0081] In some embodiments, the cpC31 integrase can mediate intra- and inter-molecular site-specific recombination at high frequency in insects. In some embodiments, transgenic insects can be created in attP-containing fly lines by integrating an attB-containing plasmid injected along with integrase mRNA into insect embryos.Xylanase preparation

[0082] In some embodiments, xylanase preparations are produced from the insects. For example, the xylanase preparations may be alive or killed insects and comprise insect eggs, larvae, pupae, adults, or any combination thereof.

[0083] Once the desired transgenic insect expressing functional xylanase is produced, a quantity of adult or larval insects is introduced into a growth environment. The growth environment may be closed or partially closed. The growth environment preferably utilises a high-volume waste source such as fish and animal waste, processed or damaged fruit, vegetable, flowers grains, plant material, or other like food sources. Other waste sources include the by-products of a processing plant utilizing animal or vegetable components fromsuch processes as farming operations, grain processing, fruit processing, or ethanol and biodiesel production.

[0084] Once an insect population has reached the desired size and quantity to maximize biomass, the insects are then separated from the introduced food source during the harvesting process. In some embodiments, insect species (such as black soldier fly) are chosen as the larvae have the ability to self-harvest. The harvesting process may include the use of vacuums, blower fans, washing stations, and screens / sieves to move and separate the insects.

[0085] The harvested insects are then dried, for example, in commercial belts and tumble driers, depending on the species and life stage (larval or adult). Once the insects have been desiccated, they may be ground into a meal.

[0086] In one embodiment, the xylanase preparation consists of or consists essentially of an insect meal or powder.

[0087] The xylanase preparation may further comprise an anti-caking agent, a preservative, and optionally a surfactant.

[0088] Additionally, the xylanase preparation in the form of a meal or powder may be used as animal feed or feed additive which may contain other substituents such as colouring agents, aroma compounds, stabilizers, vitamins, minerals, other feed or food enhancing enzymes etc. A typical livestock feed or feed additive usually comprises one or more compounds such as vitamins, minerals or feed enhancing enzymes and suitable carriers and / or excipients.Uses

[0089] Processing of plant derived non-starch polysaccharides (NSPs) found in cereal grains (e.g., wheat, barley or corn) require carbohydrase enzymes that depolymerize xylan. Xylanase enzymes are capable of hydrolysing glycosidic linkages on xylan and convert it into smaller oligosaccharides that are easier to process. The xylanase preparations described herein may be used as animal feed, supplements, feed additives or as part of a feed additive to increase the nutritional value and / or improve digestibility of animal feed for livestock animals such as pigs, chickens, cattle, cows, goats, sheep, shellfish, fish, etc., or domestic pets such as dogs, cats etc. The livestock animal may be a monogastric animal such as a pig or chicken.

[0090] In some embodiments, the xylanase preparation described herein increases the metabolizable or net energy of raw ingredients and therefore increases the totalmetabolizable or net energy content of the diet. Zootechnical performances can therefore be maintained with less raw energy in less expensive diets (i.e., less fat / oil and more fiber).

[0091] In some embodiments, the xylanase preparation described herein increases the release of (micro) nutrients entrapped within the cell walls of the feed. Such entrapment is due to the presence of non-starch polysaccharides that are resistant to the digestion by the animal.

[0092] In some embodiments, the xylanase preparation described herein increases the efficiency of digestion and / or assimilation of nutrients.

[0093] In some embodiments, the xylanase preparation described herein reduces digesta viscosity.

[0094] It is contemplated that the observed improved digestion will give more energy to the animal and thus increase weight gain. An increased weight gain means an improved or increased daily, weekly, bi-weekly, or monthly weight gain (in g or kg per the relevant time period), relative to a control without added xylanase.

[0095] The xylanase preparation described herein improves the apparent digestibility of the feed (e.g., as compared to a control without xylanase). Digestibility can be measured and determined by those skilled in the art using well-known methods. For example, many commercial forage testing laboratories offer in-vitro digestion assays based on techniques developed and as described previously (Goering, H. K., and P. J. Van Soest. 1970. Forage Fiber Analyses (Apparatus, Reagents, Procedures, and Some Applications). Agric. Handbook No. 379. ARS-USDA, Washington, DC.).

[0096] A "feed", "livestock feed", "animal feed" or the like means any natural or artificial diet, meal or the like or components of such meals intended or suitable for being eaten, taken in, digested, by an animal including a human being. The term "feed additive" as used herein, denotes, for example, a composition containing agents that provide a beneficial effect to an animal (e.g., a therapeutic or digestive effect). A "feed additive" typically is not a source of caloric intake for an animal, in other words, a feed additive typically is not a source of energy for the animal, but rather is a composition which is taken in addition to typical "feed" or "animal feed".

[0097] The xylanase preparations is supplemented to the livestock animal, e.g., fish or chicken, before or simultaneously with the feed / diet. In one embodiment, the xylanase preparation of the invention is supplemented to the animal simultaneously with the feed / diet. In another embodiment, the xylanase preparation is added to the feed / diet in the form of a granulate or a stabilized liquid.

[0098] In some embodiments, the xylanase preparation described herein is used for other industrial applications such as preprocessing NSPs as feedstocks for ethanol production or microbial bioreactors.

[0099] 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.ExamplesExample 1 : Materials and Methods1.01 Plasmid assembly

[0100] Synthetic sequences containing genes related to two xylanases from Trichoderma reesei as previously described (Torronen, A., et aL, Biotechnology, 1992. 10(11 ): p.1461 - 1465) were synthesised as gBLOCKs (integrated DNA technologies, IDT). The sequences contained short 3' and 5' homology arms, Kozack consensus sequence AAAAA, and a larval cuticle protein 9 signal peptide sequence upstream from the xylanase gene. The sequences also contained start and stop codons and were codon optimised for expression in Drosophila melanogaster using the optimisation tool provided by IDT (https: / / sg.idtdna.com / CodonOpt). The sequences were inserted into a plasmid containing a short a-tubulin promoter and a downstream SV40 polyadenylation termination sequence (pMC-1-1-1) using HiFi assembly (NEB catalogue). This backbone was chosen as it had previously showed stable enzyme expression (Clark, M., et aL, ACS Synthetic Biology, 2021. 11(1 ): p. 308-316). Two xylanase constructs were assembled, one containing a native propeptide region (Xylan2-1) and the other without a propeptide region (Xylan2-2). Once assembled, the plasmids were digested using Ncol and Ndel to confirm correct assembly. Sanger sequencing was performed at the Garvin institute of medical research (NSW, Australia). The correct plasmids were then sent to BestGene Inc. (Chino Hills, USA) for cpC31 -mediated integration.Table 1. Sanger sequencing primers1.02 Generation of transgenic insects

[0101] The generation of transgenic D. melanogaster was outsourced to BestGene Inc. (Chino Hills, CA) for cpC31 -mediated integration. For this study, the specific package chosen was Plan I which involves transgene integration with white eyed transformants (w+) along with balancer lines. The BDSC#25709 strain of D. melanogaster was used to allow integration of the transgene on the 2ndchromosome. The balancer gene, identified as CyO, exhibited "Curly wings" phenotype, enabling the differentiation of homozygous individuals. Homozygous lines were established and maintained on Bloomington fly diet ad libitum which was changed monthly in controlled environment rooms set to 25°C, 75% humidity, 12 / 12hr light / dark cycle.1.03 Fly lysate preparation

[0102] Only homozygous flies were selected for experiments. 50 adult transgenic individuals were collected from each strain and frozen at -30°C for at least 12 hours (Thermo scientific TSX freezer). These individuals were then homogenised to make a crude lysate using microtube pellet pestle (Kontes) in 500 pL of a 100 mM sodium acetate extraction buffer containing 0.5 mg / mL bovine serum albumin at pH 4.5 and kept on ice. Samples were then centrifuged at 16,000x g for 10 minutes in a refrigerated benchtop centrifuge (Sigma, 1 -14K centrifuge) maintained at 4°C to obtain the supernatant.1.04 Xylanase assay

[0103] The activity of the xylanase enzymes in D. melanogaster was determined using a xylanase assay kit produced by Megazyme. The flies were processed as outlined above and prior to the start of an assay, the supernatant was transferred to a clean Eppendorf tube and warmed to room temperature for use. This supernatant was then preincubated at 40°C for 20 minutes on a thermoblock (HB-R, Daihan scientific) and spun down on a benchtop centrifuge (Pico 17 microcentrifuge, Thermofisher Scientific) at 16,000x g for 5 minutes. After this centrifugation, a supernatant was taken, and this final sample was used for in-vitro assays.

[0104] Assays to test for enzyme function were performed using a Xylanase assay kit (XylX6 method) from Megazyme (product code: K-XylX6-2V, NeoGen corp.) with an altered method as follows. As the amount of xylanase being produced by transgenic D.melanogaster was uncertain and due to the small volumes of lysate material, the volumes of the assay kit were reduced to fit into a 96 well microplate (Greiner Bio-One, catalogue: 655101) to be analysed by a microplate reader (Spectrostar nano, BMG Labtech). A large proportion of the well content was suggested to be the stopping reagent, the effectiveness of different Tris concentrations was tested. This involved running the assay with different concentrations of stopping buffer to assess any interactions with the enzyme. Some interaction was observed and it was determined that 100 pL of 6% Tris buffer at pH 10 would adequately stop the reaction. The assay absorbance was read at 400 nm and multiple time points were analysed, including timepoints 0 minutes, 20 minutes, 30 minutes, 45 minutes, and 1 hour. Samples assayed included the same number of flies from an empty vector strain, a reagent blank, a positive control provided by the XylX6 assay kit and the Xylan2-1 and Xylan2-2 samples.

[0105] To test for enzyme activity, the XylX6 assay was performed with a final well content of 10 pL of substrate, 210 pL of lysate sample as described above, and 100 pL of Tris stopping buffer. Due to the possibly dilute nature of the xylanase in the transgenic D. melanogaster samples, these assays were allowed to incubate for 0, 10, 20, 30, 60, 90 and 120 minutes. After the addition of the Tris buffer samples were centrifuged in a benchtop centrifuge at 16,000x g for 5 minutes and the supernatant was added to the 96 well plate to ensure that no crashed-out protein could affect the absorbance readings of the microplate reader.1.05 Calculation of enzyme activity

[0106] Enzyme activity was calculated following instructions from the xylanase assay kit, where it is defined that one unit of xylanase activity represents the amount of enzyme required to release one micromole of 4-nitrophenol from the xylan substrate.Ai4400 Total volume in cell 1 Extraction volume— X X XIncubation time Aliquot assayed smM Sample volume

[0107] Where AA400 represents the absorbance of the sample minus the absorbance of the blank, and cmM represents the absorbance production of 4-nitrophenol of the Tris stopping buffer at pH 10. Enzyme activity for an individual fly was then determined by dividing the result of this equation by the number of flies in the sample.1.06 Animal rearing methods

[0108] Both wild type and experimental strains of D. melanogaster were reared in polystyrene vials (Flystuff, narrow Drosophila vials, #32-109RL) with a standard cornmeal diet (for 1 L: 920mL water, 67g cornmeal, 4.6g agar, 50g sugar, 15.88g yeast, 9.18g soyflour, 60mL of 1 M propionic acid). Flies are transferred to new vials approximately every two weeks. Fly sorting and selection was done by anaesthetising the flies with carbon dioxide before transferral to a Flypad (Flystuff, Genesee Scientific) with additional carbon dioxide being released from the Flypad in order to keep the flies anaesthetised. Observation for the selection of homozygosity was performed under a microscope (Leica MZ 6). Flies were secured in a controlled environment room at 25°C, approximately 60% humidity, and a 12 hour light / dark cycle under LED lights with a 30 minute dusk / dawn transition period.Disposal of flies was carried out by freezing the flies in their bottles in a -30°C freezer for at least 48 hours before being autoclaved.1.07 Xylanase- Trichoderma reesei

[0109] The transgenic strains expressed two different xylanases from Trichoderma reesei, 'Xylan2-1' containing a native propeptide region (SEQ ID NO: 1) or 'Xylan2-2' without a propeptide region (SEQ D NO: 2). In both constructs, the endogenous signal peptide was replaced by the D. melanogaster larval cuticle protein 9 and the sequence was codon optimized for D. melanogaster. Expression was driven by a short variant of the D. melanogaster tubulin promoter. The plasmids used to generate the transgenic flies were designated pJM2-1-1_Xylan for Xylan2-1 (SEQ ID NO: 3 which comprise SEQ ID NO: 1) or pJM2-2-1 Xylan for Xylan2-2 (SEQ ID NO: 4 which comprise SEQ ID NO: 2).1.08 Statistical analysis

[0110] All statistical analysis were performed using R studio (Version 2023.09.1 build 494). Samples were compared using one-way ANOVA and Tukey HSD post-hoc test. All samples were timepoint 0 minutes subtracted to account for shifts in absorbance.Example 2: Results and Discussion

[0111] Results from the in-vitro assays show a significant shift of the Xylan2-1 sample towards 400nm, illustrating the activity of endo-1 ,4-p-xylanase in transgenic D. melanogaster. Additionally, the Xylan2-1 construct shows a greater increase in absorbance compared to the Xylan2-2 construct, suggesting that the inclusion of a propeptide region increases the ability of the xylanase to cleave glycosidic linkages and function. This result was consistent in a number of trials even with several different strains of D. melanogaster. As an adult D. melanogaster can lay up to 50 eggs per day per female and has a generation time of roughly 10 days this illustrates that insects can be used effectively to mass produce high value bioproducts.

[0112] The production of functional xylanases in insects represents a change to the production of microbial enzymes that benefits new producers of these importantbiomolecules. Previous cost analyses of enzyme production have shown that a contributing factor to the costs associated with producing enzymes, are the cost of the substrate (28% of total costs). Adding to this, further costs are associated with upstream and downstream processing of material that is related to the extraction of the enzymes from substrate or microbial hosts. As some insect species can be grown effectively on municipal or agricultural wastes, this could reduce costs by adopting insects as host organisms. This is particularly relevant for the animal feed industry as insects can increase palatability and digestibility of feed for livestock animals including fish and poultry.

Claims

Claims:1 . A transgenic insect capable of expressing an active fungal xylanase, the insect comprising a heterologous nucleic acid encoding the fungal xylanase, wherein the heterologous nucleic acid is integrated in the insect genome, and wherein the heterologous nucleic acid is operably linked to a promoter.

2. The transgenic insect of claim 1 , wherein the fungal xylanase is from Trichoderma reesei.

3. The transgenic insect of claim 1 or 2, wherein the heterologous nucleic acid comprises SEQ ID NO: 1 or a sequence at least 80%, 85%, 90%, 95%, 97%, or 99% identical to SEQ ID NO: 1 .

4. The transgenic insect of claim 1 or 2, wherein the heterologous nucleic acid comprises SEQ ID NO: 2 or a sequence at least 80%, 85%, 90%, 95%, 97%, or 99% identical to SEQ ID NO: 2.

5. The transgenic insect of any one of claims 1 to 4, wherein the heterologous nucleic acid further comprises a sequence encoding a signal peptide in frame with the sequence encoding the fungal xylanase.

6. The transgenic insect of claim 5, wherein the signal peptide is the larval cuticle protein 9 signal peptide or a sequence at least 80%, 85%, 90%, 95%, 97%, or 99% identical to larval cuticle protein 9.

7. The transgenic insect of any one of claims 1 to 6, wherein the promoter is the short tubulin alpha promoter.

8. The transgenic insect of any one of claims 1 to 7, wherein the insect is selected from the genus Hermetia, Drosophila, or Tenebrio.

9. The transgenic insect of claim 8, wherein the insect is Hermetia illucens, Drosophila melanogaster or Tenebrio molitor.

10. A xylanase preparation comprising the transgenic insect of any one of claims 1 to 9, or a portion thereof.11 . The xylanase preparation of claim 10, wherein the transgenic insect is fully or partially dried.

12. The xylanase preparation of claim 10 or 11 , wherein the transgenic insect is in the form of a meal or a powder.

13. The xylanase preparation of any one of claims 10 to 12, further comprising one or more of a preservative, anti-caking agent or surfactant.

14. A feed additive composition comprising a xylanase preparation of any one of claims 10 to 13.

15. A method for improving nutrient availability or digestibility of an animal feed comprising combining an effective amount of a xylanase preparation of any one of claims 10 to 13 or a feed additive composition of claim 14 with the animal feed.

16. The method of claim 15, further comprising combining at least one additional ingredient selected from vitamins and minerals.

17. The method of claim 15 or 15, wherein the animal is selected from the group consisting of chicken, pig, cattle, sheep, goat, shellfish and fish.

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