Transgenic insects and preparations thereof

Abstract

There is provided a transgenic insect comprising a heterologous nucleic acid encoding a bacterial α-amylase, 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 α-amylase allows for the production of an active bacterial α-amylase in the insect. The insect or α-amylase 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. 2023903730 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 bacterial a-amylase and use of the transgenic insects or a- amylase preparation made from the insects for animal feed or feed additives and other industrial applications.Background

[0003] Amylases enzymes are often used in industrial processing including in the food, fermentation, paper, textile, and pharmaceutical industries. In particular, amylases are used as direct supplements in animal feed, in paper pulp bleaching, in the bioconversion of agrowastes and lignocellulosic material into formative products, and to clarify brewed beverages. Starch is a primary component in many animal diets and is a major storage product of economically important crops such as wheat, rice, maize, tapioca, and potato. The industrial processing of starch requires enzymatic breakdown by a-amylases which can be obtained from plants and animals but is most commonly extracted from microbial and fungal sources. However, few species possess a-amylases with desirable qualities such as thermostability or pH stability, and as such the commercial production of a-amylases is predominantly from Bacillus stearothermophilus, Bacillus amyloliquefaciens, and Bacillus licheniformis cultivated in solid-state fermentation systems.

[0004] a-Amylase (a-1 ,4-glucan-4-glucanohydrolase) catalyzes the hydrolysis of internal a- 1 ,4-glycosidic linkages in starch into low molecular weight products such as glucose, maltose and maltotriose units. Supplementation with a-amylases in animal feed improves digestibility and energy value of the feed by degrading starch and releasing more simple sugars. Commercially purified a-amylases have been produced for this purpose via microbial fermentation which requires sophisticated and expensive infrastructure to purify the enzymes.

[0005] Accordingly, there is a need for methods of producing a-amylases in an efficient and cost-effective manner with comparably small infrastructure requirements.

[0006] The present inventors have developed methods of producing bacterial a-amylases in transgenic insects. The methods produce insects that generate value from organic waste as the insects, such as black soldier flies, are able to survive and proliferate on a diet of organic waste. The insects can also be used as livestock feed or feed ingredients. The methods significantly reduce resource and time required for producing a-amylases for feed additives. The a-amylase preparation made from insects can be used for other industrial processes.Summary

[0007] The invention generally relates to a method of producing functional bacterial a- amylase in transgenic insects. The invention also relates to a method of improving nutrient availability and / or digestibility of an animal feed by combining an effective amount of a- amylase prepared from the transgenic insects with the animal feed.

[0008] In a first aspect, the invention relates to a transgenic insect capable of expressing an active bacterial a-amylase, the insect comprising a heterologous nucleic acid encoding the bacterial a-amylase, wherein the heterologous nucleic acid is integrated in the insect genome, and wherein the heterologous nucleic acid is operably linked to a promoter. The bacterial a-amylase may be thermostable, for example the thermostable bacterial a- amylase may retain activity after exposure to a temperature of 60°C.

[0009] The bacterial a-amylase may be from Bacillus licheniformis.

[0010] 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 .

[0011] The heterologous nucleic acid may further comprise a sequence encoding a signal peptide in frame with the sequence encoding the bacterial a-amylase. 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.

[0012] The promoter may be a short tubulin alpha promoter.

[0013] 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.

[0014] In a second aspect, the invention relates to an a-amylase preparation comprising the transgenic insect of the first aspect, or a portion thereof.

[0015] The transgenic insect of the a-amylase preparation may be fully or partially dried. The transgenic insect of the a-amylase preparation may be in the form of a meal or apowder. The a-amylase preparation may further comprise one or more of a preservative, anti-caking agent or surfactant.

[0016] In a third aspect, the invention relates to a feed additive composition comprising an a-amylase preparation of the second aspect.

[0017] 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 an a-amylase 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.

[0018] The animal may be, for example, chicken, pig, cattle, sheep, goat, and fish.Definitions

[0019] 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.

[0020] 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.

[0021] Throughout this specification, the term 'consisting of' means consisting only of.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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 an a-amylase preparation or feed additive composition as described herein, wherein the specified parameter is elevated as compared with an animal not fed a-amylase or an a- amylase preparation or feed additive composition as described herein, for example, is fed a conventional feed that does not comprise exogenous a-amylase.

[0029] 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.

[0030] 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

[0031] Figure 1 : Thermal tolerance of Bacillus licheniformis a-amylase produced by transgenic insect was determined by preincubating the supernatant of transgenic insect lysates at different temperatures 40, 60, 70, 80 or 100°C for 20 minutes and determining the enzyme activity. Timepoint 0 subtracted data showing absorbance change of Amyl 2-1 samples after different preincubation temperatures are shown. Data on samples that had been stored frozen but not preincubated at high temperatures (-20 values) are also shown to determine how the enzyme performs in ambient temperature. All samples were left to react with the substrate for 10 minutes before stopping reagent was added. Amyl 2-1 = transgenic Drosophila melanogaster; EV = empty vector; Pos = positive control extracted from Malt flour; RB = reagent blank. Samples with shifts in absorbance less than 0.15 are considered instrument noise. a-Amylase activity was determined via commercially available a-amylase assay kit (Megazyme, Ireland) and determined via UV-vis spectrometry at 400nm.

[0032] Figure 2: Bacillus licheniformis a-amylase activity following preincubation at 40°C for 20 minutes and at 60°C for 20 minutes. Data show changes in the absorbance of samples over time (0, 5, 10, and 15 minutes). Dotted line indicates samples that were preincubated at 60°C; solid line indicates samples that were preincubated at 40°C. Amyl 2- 1 = transgenic Drosophila melanogaster; EV = empty vector; Pos = positive control extracted from Malt flour; RB = reagent blank. Change in absorbance less than 0.15 is considered instrument noise. a-Amylase activity was determined via commercially available a-amylase assay kit (Megazyme, Ireland) and determined via UV-vis spectrometry at 400nm.Description of Embodiments

[0033] The technology described herein generally relates to transgenic insects expressing an a-amylase enzyme. The insect, preferably in the larval or pupal stage, is used as the base for an a-amylase preparation containing the expressed a-amylase. The a-amylase preparation can be used as a feed additive for livestock animals to improve nutrient availability or digestibility of an animal feed.

[0034] Described herein are insects (e.g., Hermetia illucens) that are genetically engineered to express a functional a-amylase from bacteria (e.g., Bacillus stearothermophilus, Bacillus amyloliquefaciens, or Bacillus licheniformis). Transgenic insects expressing a functional a-amylase are capable of hydrolysing internal a-1 ,4- glycosidic linkages in starch into low molecular weight products such as glucose and can be used to improve nutrient availability or digestibility of an animal feed. The use of transgenicinsects expressing a-amylase as animal feed or additive can increase the energy value of the feed by degrading starch and releasing simple sugars. a-Amylase

[0035] In the context of this disclosure, a-amylase (systematical name: 1 ,4-alpha-D-glucan glucanohydrolase) refers to the enzyme classification E.C.3.2.1 .1 . a-Amylases act on, for example, starch, glycogen and related polysaccharides and oligosaccharides in a random manner, liberating reducing groups in the alpha configuration. In a preferred embodiment, the a-amylase is thermostable. In another preferred embodiment, the a-amylase is pH stable (e.g., an acid pH). Even more preferably, the a-amylase is thermostable and pH stable.

[0036] Amylase activity may be determined by any suitable assay. Generally, assay-pH and assay-temperature may be adapted to the enzyme. Amylases may be of bacterial or fungal origin. It is contemplated that any amylase known in the art may be used in the recombinant insects described herein and a skilled person will be able to identify suitable amylases.

[0037] Microbial amylase enzymes may be derived from bacteria or fungi. Suitable bacterial amylase enzymes include but is not limited to Bacillus amyloliquefaciens, Bacillus circulans, Bacillus halmapalus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, and Bacillus subtilis. Preferably, the bacterial amylase is from the genus Bacillus. Suitable fungal amylase enzymes include but is not limited to Aspergillus oryzae, Aspergillus niger, Aspergillus phoenicis, Aspergillus awamori, Rhizopus microspores, Penicillium brevicompactum, and Penicillium chrysogenum. Preferably the fungal amylase is from filamentous fungi, more preferably from the genus Aspergillus. In one embodiment, the amylase enzyme is derived from yeast, for example, Saccharomycopsis fibuligera. In one embodiment, the amylase enzyme is derived from archaea, for example, Pyrococcus furiosus.

[0038] In one embodiment preferably the amylase, e.g., a-amylase, is from Bacillus stearothermophilus, Bacillus amyloliquefaciens, or Bacillus licheniformis.

[0039] The a-amylase may contain various sequence changes from the wild-type or naturally occurring a-amylase. In this context, the term "% identity" refers to the level of nucleic acid or amino acid sequence identity between the modified a-amylase and the wildtype a-amylase. For example, modified a-amylase 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 a-amylase.

[0040] In one embodiment, the a-amylase is a Bacillus licheniformis a-amylase, for example, a Bacillus licheniformis a-amylase encoded by SEQ ID NO: 1 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.

[0041] In some embodiments, it may be desirable to modify the a-amylase. 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, PCR 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 a-amylase) and codon optimisation for generating high expression constructs.

[0042] In some embodiments, the nucleic acids encoding the a-amylase 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 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.

[0043] 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.

[0044] 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).

[0045] A skilled person will recognise that other modifications can be made to the a- amylase 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.

[0046] In some embodiments, the amylase is not a thermostable amylase. In some embodiments, the amylase is a thermostable amylase. In some embodiments, the amylase can tolerate a wide pH range (e.g., is active across a wide pH range, including acidic pH).

[0047] Manipulating the properties of the amylase 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 amylases.

[0048] In order to express a-amylase in an insect, a nucleic acid encoding the a-amylase 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 a-amylase and optionally other sequences such as a transcription terminator and a sequence encoding a signal peptide.

[0049] The promoter can be constitutive or inducible. Inducible promoters can be advantageous because the insect cells can be grown to high densities before the a-amylase polypeptide expression is induced.

[0050] Examples of suitable constitutive promoters are actin 5C promoter, 5'LTR of a COPIA element, or the short tubulin alpha promoter.

[0051] In one embodiment, the short tubulin alpha promoter is used.

[0052] 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.

[0053] Suitable inducible promoters include metallothionen, HSp-70 promoter, and tetracycline operator promoters.

[0054] In some embodiments, the a-amylase is secreted. Amylases 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 amylase, or the nucleic acid sequence encoding the amylase signal sequence may be replaced by a nucleic acid sequence encoding a heterologous signal peptide.

[0055] Numerous signal peptides are known in the art and include signal peptides from multiple sources such as signal peptides from human beta-interferon, human placental alkaline 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 amylase 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.

[0056] In one embodiment, the signal peptide is from larval cuticle protein 9.

[0057] In other embodiments, the a-amylase is expressed intracellularly and in these embodiments the a-amylase in not fused to a signal peptide.Insects

[0058] One advantage of the present invention is that a-amylase 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 a-amylase.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 a-amylase and form the basis of an a-amylase 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 a- amylase) production may be made by the skilled person.

[0059] 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 starch, glycogen and related polysaccharides and oligosaccharides, to increase growth.

[0060] 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.

[0061] 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 a- amylase 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).

[0062] It is envisaged that any dipteran insect may be used to express the a-amylase. Suitable dipteran insects include soldier flies, robber flies, bee flies, hover flies, fruit flies, vinegar flies, and blowflies.

[0063] 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.

[0064] In other embodiments, the insect may be a mealworm, for example, of the genus Tenebrio. In one embodiment, the mealworm is Tenebrio molitor.

[0065] The a-amylase can be expressed in one or any combination of the eggs, larvae, pupae, and adults.Transformation of Insects

[0066] In general, transformation is a process in which exogenous DNA sequences are introduced into the insect germ line. Any amylase 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.

[0067] To produce heritable changes, the a-amylase or expression vector containing the a- amylase must be transformed into the insect during early development, prior to germ cell formation, 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.

[0068] 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.

[0069] Microinjection can be performed as "co-injection” or “direct” injections. During “coinjection,” two components are introduced: the amylase 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.

[0070] 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.

[0071] 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 means that 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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, the frequency 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.

[0076] 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.

[0077] 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 reactionoccurs. It has been demonstrated that the integrase can recognize and integrate into endogenous pseudo attP sites that have partial identity to attP.

[0078] 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.Amylase preparation

[0079] In some embodiments, a-amylase preparations are produced from the insects. For example, the a-amylase preparations may be alive or killed insects and comprise insect eggs, larvae, pupae, adults, or any combination thereof.

[0080] Once the desired transgenic insect expressing functional a-amylase 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 from such processes as farming operations, grain processing, fruit processing, or ethanol and biodiesel production.

[0081] 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.

[0082] 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.

[0083] In one embodiment, the a-amylase preparation consists of or consists essentially of an insect meal or powder.

[0084] The a-amylase preparation may further comprise an anti-caking agent, a preservative, and optionally a surfactant.

[0085] Additionally, the a-amylase 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 colouringagents, 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

[0086] Amylase enzymes are capable of hydrolysing starch to shorter-chain oligosaccharides such as maltose. The glucose moiety can then be more easily transferred from maltose to a monoglyceride or glycosylmonoglyceride than from the original starch molecule. The a-amylase 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, fish, etc., or domestic pets such as dogs, cats etc. The livestock animal may be a monogastric animal such as a pig or chicken.

[0087] In some embodiments, the a-amylase preparation described herein improves the total tract digestibility of crude fibre, crude protein, organic matter, and / or crude fat present in an animal feed (e.g., as compared to an animal feed without a-amylase).

[0088] In some embodiments, the a-amylase preparation described herein improves the degradation in the rumen of dietary starch, particularly slowly degradable starch such as maize starch that is not heat-treated and / or contains large particles, or potato starch, thereby contributing more energy to the rumen microorganisms and to the ruminant (e.g., cattle, sheep, and goat) itself in the form of short-chain fatty acids.

[0089] In some embodiments, the a-amylase preparation described herein improves the degradation in the small intestines of by-pass starch, i.e., starch which passes the rumen and reaches the small intestines and / or increase the glucose absorption, thus salvaging energy by minimising microbial degradation in the large intestine and excretion of starch in the faeces.

[0090] It is contemplated that the observed improved degradation of starch 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 amylase.

[0091] The a-amylase preparation described herein improves the apparent digestibility of the feed (e.g., as compared to a control without amylase). In some embodiments, the a- amylase preparation described herein improves dry matter digestibility, neutral-detergent fibre digestibility, and / or organic matter digestibility. In some embodiments, the a-amylasepreparation described herein improves starch digestibility and / or rude protein digestibility. 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.).

[0092] 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".

[0093] The a-amylase preparations is supplemented to the livestock animal, e.g., fish or chicken, before or simultaneously with the feed / diet. In one embodiment, the a-amylase preparation of the invention is supplemented to the animal simultaneously with the feed / diet. In another embodiment, the a-amylase preparation is added to the feed / diet in the form of a granulate or a stabilized liquid.

[0094] In some embodiments, the a-amylase preparation described herein is used for other industrial applications such as preprocessing feedstocks for ethanol production.

[0095] 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

[0096] A gene related to a thermostable Bacillus licheniformis a-amylase was located using the UniProt database which was originally described in Yuukt et al (Yuukt, T., et aL, The Journal of Biochemistry, 1985. 98(5): p. 1147-1156). The a-amylase sequence was ordered as a synthetic reconstruction which included a 5' homology arm, the kozak sequence AAAAA, signal peptide taken from D. melanogaster cuticle protein 9 and a 3' homology arm.This synthetic sequence was codon optimised for expression in D. melanogaster and assembled into a plasmid using Hi Fi assembly (NEBuilder Hi Fi DNA Assembly Master Mix NEB#E2621) consisting of a short a-tubulin promoter and a SV40 polyadenylation termination sequence downstream from the insertion point (plasmid name: pJM2-1 - 1_Aamyl). Once assembled, the plasmid was digested using Ncol and Ndel to confirm its 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

[0097] The generation of transgenic D. melanogaster was performed at BestGene Inc. (Chino Hills, USA) for cpC31 -mediated integration. Within this research, Plan I was specifically selected, involving the integration of transgenes with white-eyed transformants (w+) along with the provision of balancer lines. The BDSC#25709 strain of D. melanogaster was utilized to facilitate the integration of the transgene on the 2ndchromosome. The balancer gene, identified as CyO, exhibited a "Curly wings" phenotype, enabling the differentiation of homozygous individuals. These homozygous individuals were maintained on Bloomington fly diet ad libitum which was changed monthly to maintain healthy fly stocks.1.03 Fly lysate preparation

[0098] Only homozygous flies were selected for experiments. 50 adult transgenic individuals including an empty vector transgenic control were frozen at -30°C for at least 24 hours (Thermo scientific TSX freezer). These individuals were then homogenised to make a crude lysate using a motorised mortar and pestle (Kontes) in an extraction buffer consisting of 1 M sodium malate, 1 M sodium chloride, 40mM calcium chloride and 0.1% sodium azide. The lysate was kept cool at 4°C and centrifuged (Sigma, 1-14K centrifuge) for 10 minutes at 15,000x g at 4°C to obtain the supernatant.1.04 a-Amylase assay

[0099] The activity of the a-amylase enzymes in D. melanogaster was determined using an a-amylase assay kit produced by Megazyme International Ireland Ltd. (CERALPHA method, Product code: K-Cera). The flies were processed as outlined above and the supernatant was preincubated at different temperatures to determine the thermal tolerance of the enzyme. The preincubation conditions involved heating the lysates to either 40, 60, 70, 80 or 100°C and letting them incubate for 20 minutes. After this preincubation step, the samples were centrifuged at 16,000x g on a benchtop centrifuge (Pico 17 microcentrifuge, Thermofisher Scientific) and assayed using the a-amylase kit in accordance with the manufacturer’s instructions. Also tested in this assay was a transgenic D. melanogaster identically engineered by cpC31 -mediated integration with an empty vector and without a transgene. Additionally, a positive control that was extracted as per the manufacturer’s instructions and a reagent blank were tested in the in-vitro assay.

[0100] It was determined that a preincubation of 60°C for 20 minutes was sufficient to denature any native a-amylases within an empty vector D. melanogaster see Figure 1 ) and as such this was the standard incubation that lysates underwent. After the initial preincubation step, each sample was exposed to a substrate of blocked p-nitrophenol maltoheptaoside which also contained a thermostable a-glucosidase (Megazyme international Ireland Ltd.) and was vortexed then incubated at 40°C and left to react. After being left to react on a thermoblock (HB-R, Daihan Scientific), the samples were aliquoted out into Eppendorf tubes containing the stopping reagent (3% tri-sodium phosphate at pH 11) at different time points, these times included timepoints 0 min, 5 min, 10 min, and 15 min. Once aliquoted, the samples were vortexed to mix the stopping reagent and centrifuged at 16,000x g on a benchtop centrifuge to ensure that any crashed-out proteins were not affecting the absorbance of the sample. These samples were pipetted into a 96 well plate (Greiner Bio-One, catalogue:655101) and the shift in absorbance towards 400nm was analysed on a microplate reader (Spectrostar nano, BMG Labtech).

[0101] Enzyme activity is defined in the manufacturer’s instructions in the a-amylase kit (Megazyme international Ireland Ltd) where one unit of enzyme activity is the amount needed to liberate one micro-mole of p-nitrophenol from the p-nitrophenol maltoheptaoside under defined assay conditions.1.05 Animal rearing methods

[0102] 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.1.06 a-Amylase- Bacillus licheniformisThe transgenic strain expressed Bacillus licheniformis a-amylase (SEQ ID NO: 1). The endogenous signal peptide was replaced by the D. melanogaster \arva\ 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 plasmid used to generate the transgenic flies was designated pJM2-1-1_Aamyl for Bacillus licheniformis a-amylase (SEQ ID NO: 2 which comprise SEQ ID NO: 1).1.07 Statistical analysis

[0103] 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

[0104] Results from the in-vitro assays demonstrate that the bacterial a-amylase produced by Drosophila melanogaster successfully causes a shift in absorbance of samples towards 400nm, thus showing active a-amylase activity. However, as the empty vector control samples also showed a-amylase activity it was determined that a preincubation step was critical to determining the amount of thermostable a-amylase being produced by transgenic strains. When determining the thermal range of transgenic D. melanogaster a-amylase the results of the in-vitro assays showed that even when incubated to 70°C for 20 minutes the lysates were still able to liberate p-nitrophenol, despite the positive control seemed to lose the ability to degrade the substrate. Additionally, even one sample of Amyl 2-1 managed to produce a slight change in absorbance at 80°C which was seen with a shift of 0.173 after timepoint 0 subtraction.

[0105] Methods to increase the heat stability of carbohydrase enzymes such as a-amylase include using lipid-based coatings to protect the enzyme. Insects contain varied lipid profilesand the production of these enzymes within an insect host could increase the stability of these enzymes when delivered in a whole individual. This application is useful to feed based industries that are already supplementing carbohydrase enzymes in animal diets such as in fish and poultry feed.

Claims

Claims:1 . A transgenic insect capable of expressing an active bacterial a-amylase, the insect comprising a heterologous nucleic acid encoding the bacterial a-amylase, 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 bacterial a-amylase is thermostable.

3. The transgenic insect of claim 2, wherein the thermostable bacterial a-amylase retains activity after exposure to a temperature of 60°C.

4. The transgenic insect of any one of claims 1 to 3, wherein the bacterial a-amylase is from Bacillus licheniformis.

5. The transgenic insect of any one of claims 1 to 4, 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 .

6. The transgenic insect of any one of claims 1 to 5, wherein the heterologous nucleic acid further comprises a sequence encoding a signal peptide in frame with the sequence encoding the bacterial a-amylase.

7. The transgenic insect of claim 6, 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.

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

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

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

11. An a-amylase preparation comprising the transgenic insect of any one of claims 1 to 10, or a portion thereof.

12. The a-amylase preparation of claim 11 , wherein the transgenic insect is fully or partially dried.

13. The a-amylase preparation of claim 11 or 12, wherein the transgenic insect is in the form of a meal or a powder.

14. The a-amylase preparation of any one of claims 11 to 13, further comprising one or more of a preservative, anti-caking agent or surfactant.

15. A feed additive composition comprising an a-amylase preparation of any one of claims 11 to 14.

16. A method for improving nutrient availability or digestibility of an animal feed comprising combining an effective amount of an a-amylase preparation of any one of claims 11 to 14 or a feed additive composition of claim 15 with the animal feed.

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

18. The method of claim 16 or 17, wherein the animal is selected from the group consisting of chicken, pig, cattle, sheep, goat, and fish.

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