WO2011124925A2

WO2011124925A2 - Enzymes

Info

Publication number: WO2011124925A2
Application number: PCT/GB2011/050699
Authority: WO
Inventors: Yannick Pauchet; Richard Ffrench-Constant
Original assignee: University Of Exeter
Priority date: 2010-04-09
Filing date: 2011-04-08
Publication date: 2011-10-13
Also published as: GB201105987D0; WO2011124925A3; GB2479462A

Abstract

There is provided a Coleoptera polysaccharide degrading enzyme comprising an amino sequence which is: a) at least about 55 % identical to one or more of SEQ ID NOs:67-71; b) at least about 45 % identical to one or more of SEQ ID NOs:143-147; c) at least about 40 % identical to one or more of SEQ ID NOs:55-61; d) at least about 65 % identical to one or more of SEQ ID NOs:1-15; e) at least about 86 % identical to one or more of SEQ ID NOs:16-24; f) at least about 81 % identical to one or more of SEQ ID NOs:26-50 or 52-54; g) at least about 98% identical to one or more of SEQ ID NOs:51 or 62-66; or an enzymatically functional fragment or variant of any of these. There is also provided a method of degrading plant material comprising exposing the material to such a polysaccharide degrading enzyme.

Description

Enzymes

Field of the invention

The invention relates to isolated enzymes useful in the degradation of plant material and in biotechnological processes such as the preparation of biofuels and refining of food products.

Background

Biotechnology is one of the fastest growing industries globally, directly affecting many different areas including food and drink production and the pharmaceutical industry. Although 'biotechnology' (or the use of organisms to produce high-value products) is effectively an ancient science, established 8000 years ago when the first beer was brewed, in the last 50 years it has become critical to many industries around the globe. The more recent development of the biotechnology sector has been promoted by rapid interdisciplinary advances in the fields of molecular biology, genetics and analytical chemistry. Enzymes, as robust and novel catalysts for applications in biotechnology, can be regarded as sustainable alternatives (both economically as well as ecologically) to harsh and toxic chemical catalysts, as they work at moderate temperatures and thereby also reduce energy consumption. At the same time, enzymes can perform reactions difficult to perform chemically, such as the cleavage of water in photosynthesis or N₂-fixation. As industrial biotechnology enables industries to deliver novel products, which cannot be produced by conventional industrial methods, or to deliver these products with a reduced economic (and, more importantly, ecological) impact, this need for novel biocatalyst tools will only increase.

Lignocellulose is the major constituent of the cell wall of vascular plants and thereby contains most of the carbon in the planet, lignin being the barrier that protects cellulose and hemicellulose from microbial attack. Although, in nature, an enormous quantity of lignocellulose material exists, only a small fraction has been given any economic value (e.g., in agriculture or in the paper and textile industries) and most is still managed as waste. Promoting the principle of sustainable and environmentally friendly development, lignocellulose can be transformed through tailored biotechnological procedures into value-added natural products for animal feed or biofuel industries. However, the hydrolysis of plant-polysaccharides is hampered by the presence of lignin and the compact architecture of the plant cell wall requires an adequate mixture of enzymes to degrade these into fermentable sugars.

Different microorganisms, including bacteria and fungi, are involved in lignin degradation but only white-rot basidiomycetes have developed strategies to efficiently depolymerise and mineralize this 'recalcitrant' heteropolymer (Martinez et al. (1996) Eur. J. Biochem. vol. 237 pp 424-32). These organisms have a nonspecific system, including oxidoreductases, low molecular mass metabolites, dehydrogenases and activated oxygen species, whose concomitant action contributes towards effectively removing the lignin barrier, thus increasing the accessibility of plant carbohydrates. The extracellular oxidoreductases, frequently referred as lignin-modifying enzymes, play an essential role in the process. These enzymes include laccases and peroxidases that catalyze the one-electron oxidation of lignin units making subsequent progress through non-enzymatic reactions possible, leading to the production of bond cleavage products. In addition, oxidases are able to produce extracellular H2O2, as peroxidase co-substrate, which also participates in lignocellulose degradation, since it is the precursor of hydroxyl radical, the strongest oxidizing agent produced by fungi through the iron-catalyzed Haber-Weiss reaction (Akamatsu et al. (1990) FEBS Lett vol. 269 pp 261-3; Guillen et al. (2000) Appl. Environ. Microbiol, vol. 66 pp 170-5). On the other hand, the complete hydrolysis of cell wall polysaccharides (cellulose and hemicellulose) to monomeric sugars requires the synergic action of multiple enzymes with different substrate specificity, including a broad number of enzymes in the category of cellulases and xylanases. In both cases, endo-enzymes are required which cleave internally the main chains, whereas exo-enzymes liberating sugars from the ends of the polysaccharide chains and glucosidases or β-xylosidases hydrolyze the sugar dimers to glucose or xylose, respectively. Most of these enzymes have been studied in fungi, such as Trichoderma, Aspergillus or Penicillium species and full genomic information about some of them is now available (Aro et al. (2005) FEMS Microbiol. Rev. vol. 29 pp 719-39). Currently, different commercial enzymatic complexes exist which are a heterogeneous mixture of the different enzymes involved in plant-cell wall degradation, mostly containing cellulases and hemicellulases. These enzyme 'cocktails' are usually produced by fungi growing on plant cell walls. Their efficiency needs to be improved to obtain good yield in the use of plant biomass as source of fermentable sugars.

Summary of the invention

According to a first aspect of the invention, there is provided a Coleoptera polysaccharide degrading enzyme comprising an amino sequence which is:

a) at least about 55% identical to one or more of SEQ ID NOs:67-71 ;

b) at least about 45% identical to one or more of SEQ ID NOs: 143-147;

c) at least about 40% identical to one or more of SEQ ID NOs:55-61 ;

d) at least about 65% identical to one or more of SEQ ID NOs: 1-15;

e) at least about 86% identical to one or more of SEQ ID NOs: 16-24;

f) at least about 81% identical to one or more of SEQ ID NOs:26-50 or 52-54;

g) at least about 98% identical to one or more of SEQ ID NOs:51 or 62-66;

or comprising an enzymatically functional fragment or variant of any of these.

It has been found that such polysaccharide degrading enzymes are expressed by the insect order Coleoptera and that the genes encoding the enzymes can be expressed by microorganisms and/or plants used in biotechnological processes. There is a strong prejudice in the art that, when enzymes are isolated from an insect, they are in reality derived from organisms existing within the insect in a symbiotic relationship. The inventors have surprisingly found that the enzymes can, in fact, be obtained from Coleoptera insects and have been shown to be insect-derived, rather that symbiont-derived. Therefore, the term "Coleoptera polysaccharide degrading enzyme" may indicate that the enzyme is obtainable from one or more Coleoptera insects. The enzyme may be isolated, for example not contained within an insect cell.

With more than one million species (more than 25% of all known species), insects are the most species-rich group on earth and, as such, they exploit every available ecological niche. Insects are both crop pests and important vectors for infectious diseases and, therefore, previous applied entomological research has focused on crop protection and vector control. Insects can exploit every foodstuff from plant material to other animals, decaying organic material, blood, stored products and also non-food resources like wood and even buildings. It is estimated that up to half of a given crop can be lost via insect damage either in the field or upon storage.

Advantageously, a method including the use of such enzymes (as outlined below) widens the range of plant biomass starting materials that may be used in, for example, biofuel production. This enables the use of starting materials which are not also required by humans and animals for food and/or of starting materials which can grow on land and in environments which cannot be used to grow vital food crops. Possible starting materials include non-food crops such as willow (grown in temperate zones), wood from other sources, as well sugar cane (grown in the tropics) and paper. Enzymes derived from insects have not, thus far, been utilised in biotechnological processes. Such utilisation is beneficial because insects are very specialised for their habitat, so that the polysaccharide degrading enzymes expressed by them are particularly efficient for promoting the reactions which they catalyse. This enables biotechnological processes to be operated at, for example, ambient temperature, thus reducing costs. Enzymes derived from organisms such as yeasts and bacteria require temperature to be maintained higher than typical ambient temperature, typically at least 37°C. Uses of the enzymes of the invention include biofuel production, where cellulases are engineered into Oily' yeasts to facilitate the breakdown of plant material/biomass for ethanol production. In temperate countries, such as the UK, this can be to process alternative sources of biomass such as willow whilst, in the tropics, this can used for crops such as sugar cane. The enzymes can also be used in a range of applications for the preparation of food, for example the use of pectinases in the clarification of fruit juice or in the treatment of waste water generated by food industries.

The term "polysaccharide degrading enzyme" indicates an enzyme which disrupts any part of a polysaccharide molecule structure and need not be an enzyme which disrupts the glycosidic bonds between saccharide monomers. Therefore, the enzyme may be a cellulase such as endo-P-l ,4-glucanase or cellulose l,4-P-cellobiosidase, a hemicellulase such as mannan endo-l ,4-P-mannosidase, a pectinase such as polygalacturonase, pectin methylesterase or a polysaccharide lyase such as rhamnogalacturonan lyase. A description of the activity of each enzyme and their uses is set out in Table 2, at the end of the description.

In one embodiment, the Coleoptera polysaccharide degrading enzyme comprises an amino sequence which is at least about 55% identical to mannan endo-l ,4-P-mannosidase SEQ ID NO:67 and/or one or more of SEQ ID NOs:68-71, as determined by BLAST sequence comparison. For example, the enzyme may have a sequence which is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% identical to at least one of those sequences. The most closely related sequences identified by BLAST sequence comparison are sequences from molluscs, which are, at most, 40% identical to any of the sequences listed as SEQ ID NOs:67-71. The inventors have found that such enzymes are obtainable from a Callosobruchus species and have an amino acid sequence at least 55% identical to one or more of SEQ ID NO:67-70, or from a Gastrophysa species and have an amino acid sequence at least 55% identical to SEQ ID NO:71. All of the amino acid sequences SEQ ID NOs:67-71 have been found to be at least 55% identical to one another.

Therefore, enzymes exemplified by SEQ ID NOs:67-71 represent a new class of enzymes in Coleoptera, with sequence identity of 55% or more between enzymes derived from different genera. It is particularly surprising in view of the fact that the known proteins having closest sequence identity (no more than 40% identical) are found in molluscs, a completely different phylum to Coleoptera.

There are some conserved sequence domains found within the sequences SEQ ID NO:67- 71. For example, the enzyme according to this embodiment of the invention may comprise the following sequence, with the figure in square brackets indicating the position of the first amino acid of the sequence in SEQ ID NO:67: [243] (G/S)(E)(F/L/W)(T/L)(T/A)(T)(T/N)(V/I)(C/S)(E/P)(K/V)(C/T)(R)(D)(H/M)(Y) (SEQ ID NO: 158)

In this representation of the sequence, each amino acid position is represented within parentheses, with the amino acids available for selection in that position being separated by a forward slash mark "/". For example, position 1 is G or S, position 2 is always E, position 3 is F, L or W, position 4 is T or L, position 5 is T or A, position 6 is always T and so on. This may be further understood with reference to the accompanying Sequence Listing. The selection of a particular amino acid at any given position is independent of the selection of a particular amino acid at any other position within the conserved sequence. Therefore, the representation of the sequence given above is a disclosure of each and every available combination of amino acids at each position.

Other conserved consensus sequences are disclosed elsewhere in this specification and the preceding explanation of the disclosure applies to the representation of these sequences also. Alternatively or additionally, the enzyme according to this embodiment of the invention may comprise the following sequence, with the figure in square brackets indicating the position of the first amino acid of the sequence in SEQ ID NO:67:

[311] (E)(F)(S/A)(T)(T/A C)( /C/I)(S/N/T)(E)(S/C/L)(H/Q)(S/D/N)(A P/S)

(S/V/A)(W/A/K)(N)(Y)(R/H/Q)(H/Y)(I/L)(Y)(E/D/N)(G/S)(G/D)(F/Y)(G/S/V)(G)(I/A) (L/M)(S/A)(W/F)(Q)(Y)( ) (SEQ ID NO: 159)

Equivalent sequences may be identified as present in other enzyme sequences using sequence analysis and alignment methods described below, within the routine ability of the skilled person. In an embodiment, an enzyme having at least 55% sequence identity to one or more of SEQ ID NOs:67-71 and comprising one or more of consensus sequences SEQ ID NOs: 158 & 159 falls within the scope of the invention. In another embodiment, the Coleoptera polysaccharide degrading enzyme comprises an amino sequence which is at least about 45% identical to rhamnogalacturonan lyase SEQ ID NO: 143 and/or one or more of SEQ ID NOs:144-147, or to one or more of the insect- derived sequences shown in Figure 6, as determined by BLAST sequence comparison. For example, the enzyme may have a sequence which is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% identical to at least one of those sequences. Each of sequences SEQ ID NOs:143-147 is no more than 40% identical to published sequences and these sequences share at least 45% sequence identity with one another. Transcripts encoding this rhamnogalacturonan lyase (PL4) were not found in the Rice weevil or in D. abbreviatus and, to date, seem to be restricted to bark beetles only (subfamily Scolytinae). Therefore, enzymes exemplified by SEQ ID NOs: 143-147 represent a new class of enzymes in Coleoptera, with sequence identity of 45% or more between the identified enzymes.

There are some conserved sequence domains found within the sequences SEQ ID NO: 143-147. For example, the enzyme according to this embodiment of the invention may comprise the following sequence, with the figure in square brackets indicating the position of the first amino acid of the sequence in SEQ ID NO: 143:

[272] (T)(G)(S/T/N/D)(D/N/E)(S/G/E)(A S/E)(I/V)(L/V/I)(S/A)(D/A)(V/A)(A E/Q) (N/T)(Q/R)(A V)(A/Q/L/E) (SEQ ID NO: 153)

Alternatively or additionally, the enzyme according to this embodiment of the invention may comprise the following sequence, with the figure in square brackets indicating the position of the first amino acid of the sequence in SEQ ID NO: 143:

[313] (T/K/N/R)(V/I)(T/S)(G)(Q)(P/K/T)(S/K/L)(A)(T/M/A)(V/I)(M/V)(LA^)

(Y/W)(D)(S/T/A) (SEQ ID NO: 154) Alternatively or additionally, the enzyme according to this embodiment of the invention may comprise the following sequence, with the figure in square brackets indicating the position of the first amino acid of the sequence in SEQ ID NO:143:

[362] ( /R/D)(V/I)(V/A)(A)(Y)(P)(T/L/V)(A)(G)(Q/L/H)(G)(S)(E/D)( /S)(L/E) (A D)(E/K/T/R)(S/T/K)(T/S) (SEQ ID NO: 155)

Alternatively or additionally, the enzyme according to this embodiment of the invention may comprise the following sequence, with the figure in square brackets indicating the position of the first amino acid of the sequence in SEQ ID NO:143:

[455] (A T/P)(G)(I/S)(W)(T/Y/K)(I/V)(K/Q/E/T)(Y)(Q/E/D)(D)(A T/E)(X)(D/N)(G) (N/S/A/G)(G/S/A T)(R)(T/I)(L)(R)(V) (SEQ ID NO: 156)

In this sequence, "X" at position 12 is any amino acid but may be, for example, any one of K, Q, P, A, R or N.

[518] (S)(N/E)(V/I)(F/Y/I)(T/A)(V/A I)(T/N/S/P)(N/S/A)(A/N)(Q/Y)(V/L/I)(V/A) (S/D/N) (SEQ ID NO: 157)

Equivalent sequences may be identified as present in other enzyme sequences using sequence analysis and alignment methods described below, within the routine ability of the skilled person. In an embodiment, an enzyme having at least 45% sequence identity to one or more of SEQ ID NOs: 143-147 and comprising one or more of the consensus sequences SEQ ID NOs: 153-157 falls within the scope of the invention.

In another embodiment, the Coleoptera polysaccharide degrading enzyme comprises an amino sequence which is at least about 40% identical to polygalacturonase SEQ ID NO:55 and/or one or more of SEQ ID NOs:56-61 , as determined by BLAST sequence comparison. For example, the enzyme may have a sequence which is at least about 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% identical to at least one of those sequences. The most closely related sequences identified by BLAST sequence comparison are bacterial sequences which are, at most, 39% identical to any of the sequences listed as SEQ ID NOs:55-61. The inventors have found that such sequences are obtainable from a Callosobruchus species and have an amino acid sequence at least 40% identical to one or more of SEQ ID NO:55-61. All of these amino acid sequences have been found to be at least 40% identical to one another. Therefore, enzymes exemplified by SEQ ID NOs:55-61 represent a new class of enzymes in Coleoptera, with sequence identity of 40% or more between enzymes. It is particularly surprising in view of the fact that the known proteins having closest sequence identity (no more than 39% identical) are found in bacteria, in a completely different phylogenetic domain to Coleoptera. There are some conserved domains found within the sequences by SEQ ID NOs:55-61 which are not found in any comparative sequences, appearing as inserts in the representation shown in Figure 3 as compared to the polygalacturonase sequences obtained from a different Coleoptera, C. tremulae. For example, the enzyme according to this embodiment of the invention may comprise the following sequence, with the figure in square brackets indicating the position of the first amino acid of the sequence in SEQ ID NO:55 :

[93] (L/E)(Y)(P)(P)(LA^/F/T)(T/D/H/N)(V/K/A G)(K/T/LA^)(L)(P)(D/S/N)(G)(Xi)(X₂) (R/I/F/V)(X₃)(L/F/V)(X₄)(F/Y)(T) (SEQ ID NO: 160)

In this sequence, Xi, X₂, X₃ and X4 are each any amino acid, with Xi being, for example K, Q, R, L or E, X₂ being, for example, R, T, V, K, I or L, X₃ being, for example, S, H, A, N or I and X4 being, for example, P, S, D, Q or A. Alternatively or additionally, the enzyme according to this embodiment of the invention may comprise the following sequence, with the figure in square brackets indicating the position of the first amino acid of the sequence in SEQ ID NO:55:

[205] (G)(I)( )(C/V/L)(Xi)(S/P)(C/V)(R)(Y/R/Q)(L/V)(H)(I/V)(T/S/K)(G/N)(V/A) (T/D/N)(I/V)(X₂)(T/S)(G) (SEQ ID NO: 161)

In this sequence, Xi and X₂ are each any amino acid, with Xi being, for example N, V, D, E or S and X₂ being, for example, S, A, D, H or R.

Alternatively or additionally, the enzyme according to this embodiment of the invention may comprise the following sequence, with the figure in square brackets indicating the position of the first amino acid of the sequence in SEQ ID NO:55 :

[228] (LV)(AA^)(L/I/F/M)(D/N/Q/K)(A)(D/W/N/G)(G/L/M/K)(X)(G/L/D/R)(K/R/N) (SEQ ID NO: 162)

In this sequence, X is any amino acid, for example K, G, R, Q, H or Y.

Equivalent sequences may be identified as present in other enzyme sequences using sequence analysis and alignment methods described below, within the routine ability of the skilled person. In an embodiment, an enzyme having at least 40% sequence identity to one or more of SEQ ID NOs:55-61 and comprising one or more of consensus sequences SEQ ID NOs: 160-162 falls within the scope of the invention.

In another embodiment, the Coleoptera polysaccharide degrading enzyme comprises an amino sequence which is at least about 65% identical to endo-P-l ,4-glucanase SEQ ID NO: l and/or one or more of SEQ ID NOs:2-15, as determined by BLAST sequence comparison. For example, the enzyme may have a sequence which is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% identical to at least one of those sequences. Related sequences have been identified in fungi and bacteria, but those enzymes possess an additional cellulose binding domain compared to the insect enzymes, positioned at the N- or the C-terminus of the enzymatic domain. Therefore, in this embodiment of the invention, the enzyme may lack a cellulose binding domain. The presence or absence of such a domain can be readily determined by the skilled person, without use of inventive skill. For example, endo-P-l ,4-glucanase enzymes in fungi all comprise a cellulose binding domain, for example as seen in CAZy accession no. ADU33286.1.

Advantages are provided by use of these insect enzymes in the industrial context, since fewer resources are required to obtain shorter amino acid sequences, whether synthetically or by use of micro-organisms. In addition, the enzyme may be able to process a wider range of substrates in the absence of a carbohydrate binding domain, since there is a reduced limitation on the types of substrate to which the enzyme can bind.

For example, the enzyme may be obtainable from a Chrysomela species and have an amino acid sequence at least 65% identical to SEQ ID NO: l and/or 2. The enzyme may be obtainable from a Gastrophysa species and have an amino acid sequence at least 65% identical to SEQ ID NO:3, or be obtainable from a Leptinotarsa species and have an amino acid sequence at least 65% identical to one or more of SEQ ID NO:4-10. The enzyme may be obtainable from a Sitophilus species and have an amino acid sequence at least 65% identical to one or more of SEQ ID NO: l l -15.

In another embodiment, the Coleoptera polysaccharide degrading enzyme comprises an amino sequence which is at least about 86% identical to cellulose l ,4-P-cellobiosidase SEQ ID NO:16 and/or one or more of SEQ ID NOs:17-24 and/or the insect-derived sequences shown in Figure 2, as determined by BLAST sequence comparison. For example, the enzyme may have a sequence which is at least about 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% identical to at least one of those sequences. Related sequences have been identified in bacteria, but those enzymes possess an additional cellulose binding domain compared to the insect enzymes, positioned at the N-terminus of the enzymatic domain. The insect enzymes also have four extra cysteine residues conserved between sequences, as compared with bacterial sequences. For example, in SEQ ID NO:16, these residues are found at positions 181 , 213, 299 and 305. Equivalent positions in SEQ ID NOs: 17-24 can be determined by sequence alignment using methods described below, within the routine ability of the skilled person. For example, the location of the cysteine residues may be observed in Figure 2. The presence of these residues enables the formation of two additional disulphide bonds, which serve to stabilise the enzyme and improve its activity over a range of temperatures and pH, by reducing the likelihood of denaturation at extremes of temperature and pH. Advantageously, therefore, such an enzyme may be utilised in industrial processes operated at both ambient temperature and at temperatures higher than this, which might be required for other components of the process to work effectively.

For example, the enzyme may be obtainable from a Chrysomela species and have an amino acid sequence at least 86% identical to one or more of SEQ ID NOs:16 or 17, or be obtainable from a Gastrophysa species and have an amino acid sequence at least 86% identical to one or more of SEQ ID NO: 18-20. The enzyme may be obtainable from a Leptinotarsa species and have an amino acid sequence at least 86% identical to one or more of SEQ ID NOs:21-23, or be obtainable from a Sitophilus species and have an amino acid sequence at least 86% identical to SEQ ID NO:24.

For example, the enzyme according to this embodiment of the invention may comprise the following sequence, with the figure in square brackets indicating the position of the first amino acid of the sequence in SEQ ID NO: 16:

[181] (C)(E/L/Q/S)(L/A G/D)(G)(Xi)(X₂)(A/TA^)(X₃)(G/K)(P)(S)(F/Y/L)(I/M)(N) (T/N/S)(F/Y)(Q/E/G)(R)(G)(P/S)(Q/E/S)(E)(S/N)(V/T)(W)(R/K)(T/A)(I/V)(P)(Q/S) (T/P/G)(T/C/I)(C) (SEQ ID NO: 163)

In this sequence, Xi is P or no amino acid. X₂ and X₃ are each any amino acid, with X₂ being, for example, S, Q, Y, G or E and X₃ being, for example, S, D, P, E, K or N.

Alternatively or additionally, the enzyme according to this embodiment of the invention may comprise the following sequence, with the figure in square brackets indicating the position of the first amino acid of the sequence in SEQ ID NO:16:

[299] (C)(V/I)(G/S/D)(P/T/A)(Y/W/H)(N/S/A K)(C) (SEQ ID NO: 164) Equivalent sequences may be identified as present in other enzyme sequences using sequence analysis and alignment methods described below, within the routine ability of the skilled person. In an embodiment, an enzyme having at least 86% sequence identity to one or more of SEQ ID NOs: 16-24 and comprising one or more of consensus sequences SEQ ID NOs: 163 & 164 falls within the scope of the invention.

In another embodiment, the Coleoptera polysaccharide degrading enzyme comprises an amino sequence which is at least about 81% identical to polygalacturonan SEQ ID NO:26 and/or one or more of SEQ ID NOs:27-50 or 52-54, or at least about 98% identical to SEQ ID NO:51 , as determined by BLAST sequence comparison. For example, the enzyme may have a sequence which is at least about 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% identical to at least one of SEQ ID NOs:26-50 or 52-54, or at least about 99% or 100% identical to SEQ ID NO:51. For example, the enzyme may be obtainable from a Chrysomela species and have an amino acid sequence at least 81 % identical to one or more of SEQ ID NOs:26-33, or be obtainable from a Gastrophysa species and have an amino acid sequence at least 81 % identical to one or more of SEQ ID NOs:34-40, or be obtainable from a Leptinotarsa species and have an amino acid sequence at least 81% identical to one or more of SEQ ID NOs:41 -50, or be obtainable from a Sitophilus species and have an amino acid sequence at least 98% identical to SEQ ID NO:51 or at least 81% identical to one or more of SEQ ID NOs:52-54. These enzymes all include 10 cysteine residues, compared to 8 cysteine residues in related fungal sequences. For example, the additional residues are found at positions 139 and 143 in SEQ ID NO:26. Equivalent positions in SEQ ID NOs:27-54 can be determined by sequence alignment using methods described below, within the routine ability of the skilled person. For example, the location of the cysteine residues may be observed in Figure 3. Again, the additional disulphide bonds which can form help to stabilise the enzyme as compared to the fungal equivalents and improve enzyme activity at extremes of temperature and pH. As discussed above, this provides advantages when the enzyme is to be utilised in an industrial process. For example, the enzyme according to this embodiment of the invention may comprise the following sequence, with the figure in square brackets indicating the position of the first amino acid of the sequence in SEQ ID NO:26:

[135] (N/F/K)(I/L)(K/L)( )(C)(P)(Xi)(F/H/A/Q)(C)(V)(X₂)(I) (SEQ ID NO: 165) In this sequence, Xi and X₂ are each any amino acid, with Xi being, for example V, T, H, M or L and X₂ being, for example, A, S, K, I or Y.

Equivalent sequences may be identified as present in other enzyme sequences using sequence analysis and alignment methods described below, within the routine ability of the skilled person. In an embodiment, an enzyme having at least 86% sequence identity to one or more of SEQ ID NOs:26-51 or 52-54 or 98% sequence identity to SEQ ID NO:51 and comprising consensus sequence SEQ ID NOs: 165 falls within the scope of the invention.

In another embodiment, the Coleoptera polysaccharide degrading enzyme comprises an amino sequence which is at least about 98% identical to pectin methylesterase SEQ ID NO:62 and/or one or more of SEQ ID NOs:63-66 and/or the insect-derived sequences shown in Figure 4, as determined by BLAST sequence comparison. Such enzymes may be obtainable from an insect in the family Curculionidae such as a Sitophilus or a Dendroctonus species.

There are some conserved domains found within the sequences by SEQ ID NOs:62-66 which are not found in any comparative sequences, appearing as inserts in the representation shown in Figure 4 as compared to the reference sequence. For example, the enzyme according to this embodiment of the invention may comprise the following sequence, with the figure in square brackets indicating the position of the first amino acid of the sequence in SEQ ID NO:62: [33] (E)(A S)(S/N/A Q)(Xi)(Y/F)(T/I/Q)(E/T/Q)(E/A T/L)(N/T/E)(YA^)(L/F)(G/Q) (G/D)(W)(S/L/E/G)(P)(E/P/Q)(S/N/E/L)(I)(X₂)(T/L/I/V)(P/SA^)(D/K/Q/E)(X₃)(P/A)(D) (Y)(T/S/I)(V)(G/K)(X₄)(G) (SEQ ID NO: 166) In this sequence, X₃ is E, V or no amino acid. Xi, X₂ and X₄ are each any amino acid, with Xi being, for example, Y, Q, N, K or R, X₂ being, for example, S, N, H, I or V and X₄ being, for example, N, S, Y, A or V.

Alternatively or additionally, the enzyme according to this embodiment of the invention may comprise the following sequence, with the figure in square brackets indicating the position of the first amino acid of the sequence in SEQ ID NO:62:

[ 127] (T/I)(T/H/Q)(G/P/I/A)(X₁)(D/A E/G)(Y)(A K/MA^)( /S)(L/T)(V)(X₂)(X₃)(X₄)(G) (E/S/T/A)(R/L/K)(Y/F)(K/Q/N)(S/E/T)(G/A)(D) (SEQ ID NO: 167)

In this sequence, Xi is any amino acid, for example, S, T, I, L or E. X₂ is N or no amino acid. X₃ is P or no amino acid. X₄ is N, E or no amino acid.

Equivalent sequences may be identified as present in other enzyme sequences using sequence analysis and alignment methods described below, within the routine ability of the skilled person. In an embodiment, an enzyme having at least 98% sequence identity to one or more of SEQ ID NOs:62-66 and comprising one or more of consensus sequences SEQ ID NOs: 166 & 167 falls within the scope of the invention.

According to a second aspect of the invention, there is provided a method of degrading plant material comprising exposing the material to at least one polysaccharide degrading enzyme according to the first aspect of the invention, e.g., having an amino acid sequence of an enzyme obtainable from an insect of the order Coleoptera, or an enzymatically functional fragment or variant of such an enzyme. The enzyme used in the method is isolated, in that it is not contained within an insect. Enzymes having an amino acid sequence of an enzyme obtainable from the families Chrysomelidae and Curculionidae are particular examples. A related method may be a method of degrading or processing pectin-containing mixtures such as fruit juices or food industry waste water. Exposing the plant or other material to the enzyme may involve, for example, directly contacting the plant material with the enzyme by incubating the material in the presence of the enzyme or passing a liquid containing the enzyme over or through the material. Alternatively or additionally, the method of the invention may comprise a step of expressing the enzyme in a micro-organism, such as a bacterium or a yeast such as an oleaginous yeast strain used in the production of biofuels. Suitable micro-organisms are further described below.

The plant or other material may, for example, be contacted with the micro-organism which may secrete the enzyme so that it contacts the plant material and degrades it. Alternatively or additionally, an extracellular growth medium of the micro-organism may be used to contact the plant material, if the micro-organism secretes the enzyme. The micro-organism may be engineered to include proteins necessary to allow secretion of the enzyme. In a further alternative (which may also be used in addition to the previous options), the plant material may be contacted with an extract of the micro-organism, for example if the enzyme is not secreted by the micro-organism so that disruption of the micro-organism (for example, by cell lysis) is required to obtain the enzyme. In a still further alternative, a plant may itself express the enzyme (for example, by expression of a recombinant gene) which may, therefore, be available for the autodegradation of plant material containing such a plant. This may be by release of the enzyme when the plant is harvested and/or after crushing, shredding or milling of the plant material. Alternatively or additionally, the enzyme may be expressed in the cytosol and be secreted from the cell into the cell surroundings, from where it can act to degrade the plant material. Such expression may be controllable using other elements, for example temperature and/or pH- dependent factors which may "switch on" expression of the enzyme at an appropriate time during processing of the plant material.

In the method according to the second aspect of the invention, the amino acid sequence of the polysaccharide degrading enzyme may correspond to any of those having GenBank Accession Numbers CAA76931.1, AAN78326.1, AAU44973.1, AAR22385.1, BAE94320.1, BAE94321.1, CAH25542.1, CAH25543.1 , CAA76930.1 , ACP18831.1 (SEQ ID NO:25), AAG35693.1 or AAW28928.1.

In the method according to the second aspect of the invention, the amino acid sequence of the polysaccharide degrading enzyme may be encoded by and expressed from any of polynucleotide sequences SEQ ID NOs:72-142 or 148-152 or a combination of any of these. These polynucleotide sequences encode for the amino acid sequences SEQ ID NOs:l -71 and 143-147, respectively, as shown in Table 1 below.

According to a third aspect of the invention, there is provided a micro-organism comprising at least one polysaccharide degrading enzyme having (i.e., comprising or consisting of) an amino acid sequence of an enzyme according to the first aspect of the invention, for example, obtainable from an insect of the order Coleoptera, and/or of an enzymatically functional fragment or variant of such an enzyme and/or a polynucleotide coding for such an enzyme and/or for an enzymatically functional fragment or variant of such an enzyme. The enzyme may have an amino acid sequence comprising at least one of SEQ ID NOs:l-71 or 143-147, or comprising at least one amino acid sequence at least about 65% identical to one or more of SEQ ID NOs:l-15, or at least about 86% identical to one or more of SEQ ID NOs:16-24 (or any of the insect-derived sequences shown in Figure 2), or at least about 81% identical to one or more of SEQ ID NOs:26-50 or 52-54, or at least about 40% identical to one or more of SEQ ID NOs:55-61 , or at least about 98% identical to one or more of SEQ ID NOs:51 or 62-66 (or any of the insect-derived sequences shown in Figure 4), or at least about 55% identical to one or more of SEQ ID NOs:67-71 , or at least about 45% identical to one or more of SEQ ID NOs: 143-147 (or any of the insect-derived sequences shown in Figure 6). The micro-organism may alternatively or additionally comprise at least one of the polynucleotide sequences SEQ ID NOs:72-142 or 148-52 or a micro-organism equivalent thereof. The term "microorganism equivalent" of a polynucleotide sequence SEQ ID NOs:72-142 or 148-152 means a polynucleotide sequence which has been codon optimised so as to enable expression of an insect polynucleotide sequence in a micro-organism host, for example a bacterium or a yeast cell. Such codon optimisation is well understood by the skilled person and "codon" tables are available, for example, online. An example of a yeast codon optimisation table can be seen at www.yeastgenome.org/community/ codon_usage.shtml (accessed on 7 April 2011). Codon usage in the beetle species described herein is shown in Tables 3 and 4 below which, therefore, provides sufficient information to enable the skilled person to target specific codons which might be altered in a polynucleotide sequence to enable optimal expression in a yeast or other microorganism. The micro-organism may be, for example, a bacterium, a fungus, a protist, an alga or an archaeon, for example, a bacterium or a yeast such as an oleaginous yeast strain used in the production of biofuels. The micro-organism may be a strain of fungus, yeast or other micro-organism comprising at least one enzyme according to the first aspect of the invention and at least one enzyme natural to the micro-organism, i.e., present prior to the introduction of the enzyme according to the first aspect of the invention and/or a polynucleotide encoding such an enzyme. An example of a suitable micro-organism is Chrysosporium lucknowense available from Dyadic International, Inc. (Florida, USA).

According to a fourth aspect of the invention, there is provided a plant comprising at least one polysaccharide degrading enzyme having (i.e., comprising or consisting of) an amino acid sequence of an enzyme according to the first aspect of the invention, for example, obtainable from an insect of the order Coleoptera and/or of an enzymatically functional fragment or variant of such an enzyme and/or a polynucleotide coding for such an enzyme and/or for an enzymatically functional fragment or variant of such an enzyme. The enzyme may have an amino acid sequence comprising at least one of SEQ ID NOs: 1 - 71 or 143-147, or comprising at least one amino acid sequence at least about 65% identical to one or more of SEQ ID NOs:l -15, or at least about 86% identical to one or more of SEQ ID NOs: 16-24 (or any of the insect-derived sequences shown in Figure 2), or at least about 81% identical to one or more of SEQ ID NOs:26-50 or 52-54, or at least about 40% identical to one or more of SEQ ID NOs:55-61 , or at least about 98% identical to one or more of SEQ ID NOs:51 or 62-66 (or any of the insect-derived sequences shown in Figure 4), or at least about 55% identical to one or more of SEQ ID NOs:67-71 , or at least 45% identical to one or more of SEQ ID NOs:143-147 (or any of the insect- derived sequences shown in Figure 6). The plant may comprise at least one of the polynucleotide sequences SEQ ID NOs:72- 142 or 148-152 or a plant equivalent thereof. The term "plant equivalent" of a polynucleotide sequence SEQ ID NOs:72-142 or 148-152 means a polynucleotide sequence which has been codon optimised so as to enable expression of an insect polynucleotide sequence in a plant host. In a plant, the enzyme may be expressed by all cells of the plant, or by cells in a limited part of the plant, for example, in a root, flower, boll or fruit, or within a subcellular compartment such as a chloroplast, mitochondrion and/or peroxisome. The plant may be, for example, sugar cane or willow. Advantageously, plants containing such polypeptides and/or polynucleotides may facilitate autodegradation of plant material containing the plant. For example, a polysaccharide degrading enzyme may be released by crushing, shredding and/or milling of the plant, or by secretion of the enzyme from the plant.

In addition, such a plant facilitates control of insect pests using R Ai technology. The plant may comprise a polynucleotide sequence SEQ ID NOs:72-142 or 148-152, a complementary sequence thereof or a portion of any of these, the polynucleotide being a DNA molecule (capable of transcription into an RNA molecule) or an RNA molecule which is useful as an RNAi molecule. Any portion of any of the polynucleotide sequences may be used, with preferred suitable portions being 80-250bp, for example 100-200bp, 120-180bp, 130-170bp, or about 90bp, lOObp, 120bp, 140bp, 160bp, 180bp, 190bp or about 200bp in length. The term "RNAi molecule" indicates a double stranded RNA molecule which, when introduced into a cell (for example, via ingestion by an insect of a plant containing the RNA) activates the RNA interference mechanisms of the cell. Expression of the gene encoding the enzyme corresponding to the polynucleotide sequence may then be reduced or "knocked-down". Baum et al. (Nature Biotechnology (2007) vol. 25 pp 1322-1326) have shown that RNAi can be used to control insect pests. It is not necessary to kill an insect to control it, as suggested by the current widespread use of fast acting, small molecule insecticides, which largely act on the insect's nervous system. In fact, it may be desirable to simply slow its growth to avoid feeding damage or the future ability of the insect to reproduce. More specifically, if the 'fitness' of the insect is reduced, the plant may be able to compensate for the damage and thereby 'grow' out of it. Baum et al. showed that the corn rootworms of their study were not killed, but their feeding capacity was so reduced that the corn plants simply grew more roots. Therefore, damage does not reach economically important levels (where the plants fall over or 'lodge' as their roots are impaired). According to a fifth aspect of the invention, there is provided a polynucleotide encoding a polypeptide according to a first aspect of the invention. For example, the polynucleotide may encode one or more of the following polypeptides:

a) a polypeptide at least about 55% identical to one or more of SEQ ID NOs:67-71 ; b) a polypeptide at least about 45% identical to one or more of SEQ ID NOs:143-147; c) a polypeptide at least about 40% identical to one or more of SEQ ID NOs:55-61; d) a polypeptide at least about 65% identical to one or more of SEQ ID NOs:l -15;

e) a polypeptide at least about 86% identical to one or more of SEQ ID NOs:l 6-24; f) a polypeptide at least about 81% identical to one or more of SEQ ID NOs:26-50 or 52-54;

g) a polypeptide at least about 98% identical to one or more of SEQ ID NOs:51 or 62- 66; or

an enzymatically functional fragment or variant of any of these

For example, the polynucleotide may comprise at least one of SEQ ID NOs: 72-142 or 148-152, which encode for amino acid sequences 1 -71 and 143-147 respectively, as shown in Table 1 below. The polynucleotide may form part of an expression vector in which the polynucleotide is operably linked to one or more expression control sequences (e.g., a promoter sequence), so as to be useful to express a polypeptide having any of SEQ ID NOs: l-71 or 143-147 in a host cell such as a micro-organism, plant or insect cell. The present invention also encompasses structural variants of the polypeptides as defined herein. As used herein, a "variant" means a polypeptide in which the amino acid sequence differs from the base sequence from which it is derived in that one or more amino acids within the sequence are substituted for other amino acids. Amino acid substitutions may be regarded as "conservative" where an amino acid is replaced with a different amino acid with broadly similar properties. Non-conservative substitutions are where amino acids are replaced with amino acids of a different type. Table 1 showing how the amino acid sequences are encoded by the nucleic acid sequences

By "conservative substitution" is meant the substitution of an amino acid by another amino acid of the same class, in which the classes are defined as follows:

Class Amino acid examples

Nonpolar: A, V, L, I, P, M, F, W

Uncharged polar: G, S, T, C, Y, N, Q

Acidic: D, E

Basic: K, R, H. As is well known to those skilled in the art, altering the primary structure of a peptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region which is critical in determining the peptide's conformation.

In the present invention, non-conservative substitutions are possible provided that these do not interrupt with the enzymatic function of the polypeptides.

Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptides. Determination of the effect of any substitution (and, indeed, of any amino acid deletion or insertion) is wholly within the routine capabilities of the skilled person, who can readily determine whether a variant polypeptide retains the DNA polymerase activity according to the invention. For example, when determining whether a variant of the polypeptide falls within the scope of the invention (i.e., is an "enzymatically functional variant"), the skilled person will determine whether the variant retains enzyme activity (i.e., polysaccharide degrading activity) which is at least about 60%, preferably at least about 70%, more preferably at least about 80%, yet more preferably about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% the activity of the non-variant polypeptide. In some cases, the variant may have enzyme activity which is greater than 100% the activity of the non-variant polypeptide, i.e., the variant may have improved enzyme activity compared to the non-variant. A variant may also be a fragment of the whole polypeptide, i.e., a fragment which retains enzyme activity which is at least about 60% the activity of the full length polypeptide. All such variants are within the scope of the invention. Activity may be measured by, for example, any standard measure such as determining catalytic activity against a range of commercially available standard substrates such as carboxymethyl-cellulose or crystalline cellulose (for cellulases), pectin (for pectin methylesterases and rhamnogalacturonan lyases), pectic acid (for polygalacturonases) or mannan (for mannan endo-l ,4- -mannosidases). For example, the time required, under comparable conditions, to degrade a certain mass of plant material by a certain amount may be determined as a measure of the level of activity.

Using the standard genetic code, further nucleic acids encoding the polypeptides may readily be conceived and manufactured by the skilled person. The nucleic acid may be DNA or RNA, and where it is a DNA molecule, it may for example comprise a cDNA or genomic DNA.

The invention encompasses variant nucleic acids encoding the polypeptides of the invention. The term "variant" in relation to a nucleic acid sequence means any substitution of, variation of, modification of, replacement of, deletion of, or addition of one or more nucleic acid(s) from or to a polynucleotide sequence, providing the resultant polypeptide sequence encoded by the polynucleotide exhibits at least the same properties as the polypeptide encoded by the basic sequence. The term therefore includes allelic variants and also includes a polynucleotide (a "probe sequence") which substantially hybridises to the polynucleotide sequence of the present invention. Such hybridisation may occur at or between low and high stringency conditions. In general terms, low stringency conditions can be defined as hybridisation in which the washing step takes place in a 0.330-0.825 M NaCl buffer solution at a temperature of about 40-48°C below the calculated or actual melting temperature (T_m) of the probe sequence (for example, about ambient laboratory temperature to about 55°C), while high stringency conditions involve a wash in a 0.0165-0.0330 M NaCl buffer solution at a temperature of about 5- 10°C below the calculated or actual T_m of the probe sequence (for example, about 65°C). The buffer solution may, for example, be SSC buffer (0.15M NaCl and 0.015M tri- sodium citrate), with the low stringency wash taking place in 3 x SSC buffer and the high stringency wash taking place in 0.1 x SSC buffer. Steps involved in hybridisation of nucleic acid sequences have been described for example in Sambrook et al. (1989; Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor).

Typically, variants have about 55% or more of the nucleotides in common with the nucleic acid sequence of the present invention, more typically 60%, 65%, 70%, 80%, 85%, or even 90%, 95%, 98% or 99% or greater sequence identity. Variant nucleic acids of the invention may be codon-optimised for expression in a particular host cell, as mentioned above in relation to the second aspect of the invention.

Enzymes according to aspects of the invention may be prepared synthetically using conventional synthesisers. Alternatively, they may be produced using recombinant DNA technology or be isolated from natural sources followed by any chemical modification, if required. In these cases, a nucleic acid encoding the chimeric protein is incorporated into a suitable expression vector, which is then used to transform a suitable host cell, such as a prokaryotic cell such as E. coli. The transformed host cells are cultured and the protein isolated therefrom. Vectors, cells and methods of this type form further aspects of the present invention.

Sequence identity between nucleotide and amino acid sequences can be determined by comparing an alignment of the sequences. When an equivalent position in the compared sequences is occupied by the same amino acid or base, then the molecules are identical at that position. Scoring an alignment as a percentage of identity is a function of the number of identical amino acids or bases at positions shared by the compared sequences. When comparing sequences, optimal alignments may require gaps to be introduced into one or more of the sequences, to take into consideration possible insertions and/or deletions in the sequences. Sequence comparison methods may employ gap penalties so that, for the same number of identical molecules in sequences being compared, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Calculation of maximum percent identity involves the production of an optimal alignment, taking into consideration gap penalties.

Suitable computer programs for carrying out sequence comparisons are widely available in the commercial and public sector. Examples include the FASTA program (Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA vol. 85 pp 2444-2448; Altschul et al, 1990, J. Mol. Biol. vol. 215 pp 403-410), ggsearch (part of the FASTA package) (Needleman & Wunsch, 1970, J. Mol. Biol. 48: 443-453), and the BLAST software. The latter is publicly available at http://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 7 April 2011) and sequence comparisons and percentage identities mentioned in this specification have been determined using this software. The FAST A program can be accessed publicly from the European Bioinformatics Institute (http://www.ebi.ac.uk/fasta) (accessed on 7 April 2011). Typically, default parameters set by the computer programs should be used when comparing sequences. The default parameters may change depending on the type and length of sequences being compared. A comparison using the FASTA program may use default parameters of Ktup = 2, Scoring matrix = Blosum50, gap = -10 and ext = -2. A comparison using the BLAST software may use the default parameters (scoring matrix = Blosum62, gap = 11 and ext = 1). As an alternative, the percentage sequence identity may be determined using the MatGAT v2.03 computer software, available from the website http://bitincka.com ledion/matgat/ (accessed on 7 April 2011). The parameters are set at Scoring Matrix = Blosum50, First Gap = 16, Extending Gap = 4 for DNA, and Scoring Matrix = Blosum62, First Gap = 12, Extending Gap = 2 for protein.

According to an sixth aspect of the invention, there is provided a method of controlling an insect pest comprising feeding to the insect a plant according to the fourth aspect of the invention, the plant comprising a polynucleotide coding for an enzyme according to the first aspect of the invention, for example, obtainable from an insect of the order Coleoptera or coding for a portion of such an enzyme. The insect is preferably of the order Coleoptera such as, by way of non-limiting example, Colorado Potato Beetle, Corn Rootworm, Coffee Berry Borer. "Controlling" the insect pest may comprise causing the death of the insect or causing reduced growth, mobility or reproductive capability (e.g., the ability to reproduce or reducing the number of progeny per generation) of the insect, or reversed or arrested development of the insect (since some insects cease to change between instars when starved, remaining as non-reproducing juveniles). The polynucleotide may be any of those disclosed in the fifth aspect of the invention, or a portion of any such polynucleotide. The polynucleotide is preferably an RNA molecule which is useful as an RNAi molecule. Any portion of the polynucleotide sequences may be used, with preferred suitable portions being 80-250bp, for example 100-200bp, 120- 180bp, 130-170bp, or about 90bp, lOObp, 120bp, 140bp, 160bp, 180bp, 190bp or about 200bp in length. Preferably, a process of RNA interference occurs in the insect after the insect has ingested the plant, double-stranded RNA coding for an enzyme obtainable from an insect of the order Coleoptera or coding for a portion of such an enzyme having been expressed in the plant.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to" and do not exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.

Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

Brief description of Figures

Embodiments of the invention will now be described, by way of example only, with reference to Figures 1 -6 in which:

Figure 1 shows the amino acid alignment of endo-P-l,4-glucanase (GH45) enzymes from beetles, with the amino acid sequence of the enzymatic domain of the endoglucanase from Humicola insolens used as a reference sequence (not including the cellulose binding domain); conserved amino acids are highlighted dark grey, partially conserved amino acids highlighted pale grey and catalytic residues (according to H. insolens) marked *; Figure 2 shows the amino acid alignment of cellulose l,4-P-cellobiosidase (GH48) enzymes from beetles, with the amino acid sequence of the enzymatic domain of the cellulose Cell48F from Clostridium cellulolyticum used as a reference sequence (not including the cellulose binding domain); conserved amino acids are highlighted dark grey, partially conserved amino acids highlighted pale grey and catalytic residues (according to C cellulolyticum) marked ;

Figure 3 shows the amino acid alignment of polygalacturonase (GH28) enzymes from C. tremulae and C. maculatus, with the amino acid sequence of the endopolygalacturonase II from Aspergillus niger used as a reference sequence; conserved amino acids are highlighted dark grey, partially conserved amino acids highlighted pale grey and catalytic residues (according to A. niger) marked *;

Figure 4 shows the amino acid alignment of pectin methylesterase (CE8) enzymes from S. oryzae, with the amino acid sequence of the pectin methylesterase from Erwinia chrysanthemi used as a reference sequence; conserved amino acids are highlighted dark grey, partially conserved amino acids highlighted pale grey and catalytic residues (according to E. chrysanthemi) marked†;

Figure 5 shows the amino acid alignment of mannan endo-l ,4- -mannosidase (GH5) enzymes from beetles, with the amino acid sequence of the β-mannanase from the blue mussel Mytilus edulis used as a reference sequence; conserved amino acids are highlighted dark grey, partially conserved amino acids highlighted pale grey and catalytic residues (according to M. edulis) marked *; and

Figure 6 shows the amino acid alignment of rhamnogalacturonan lyase (PL4) enzymes from beetles, with the amino acid sequence of the RhiE protein from the Erwinia chrysanthemi bacterium used as a reference sequence; conserved amino acids are highlighted dark grey and partially conserved amino acids highlighted pale grey. Examples

Insect selection

Six key species from within the two groups Chrysomelidae (leaf beetles) and Curculionidae (weevils) were selected. Species were chosen based upon pest status and differences in their feeding behaviour (e.g., fresh plant tissue versus stored product feeder). The selected species are:

(1) The Poplar leaf beetle, Chrysomela tremulae (Chrysomelidae);

(2) The Green dock beetle, Gastrophysa viridula (Chrysomelidae);

(3) The Colorado potato beetle, Leptinotarsa decemlineata (Chrysomelidae);

(4) The Cowpea bruchid, Callosobruchus maculatus (Chrysomelidae);

(5) The Rice weevil, Sitophilus oryzae (Curculionidae); and

(6) The Mountain Pine Beetle, Dendroctonus ponderosae (Curculionidae)

454 sequencing of beetle midgut transcriptomes

Actively feeding late instar larvae were dissected from all six beetle species. In the case of the large leaf feeding beetles, this was performed on larvae collected from laboratory cultures. For the insects whose larvae feed inside of pulses or grains, the grains were physically cracked and sieved to remove actively feeding larvae which were then dissected, midguts washed and stored at -80°C before R A isolation. To prevent over- representation of the most common transcripts and, therefore, to enhance the overall rate of gene discovery, the resulting double-stranded cDNAs were 'normalised' using the Kamchatka crab duplex-specific nuclease method (Zhulidov et al. Nucl. Ac. Res. (2004) vol. 32 e37).

Trimming and assembly of the raw sequence data was achieved using ⁱest2assembly' (Papanicolaou et al. BMC Bioinformatics (2010) vol. 10:447). This program trims sequencing adaptors from the raw sequence reads and then assembles them using two different assembly algorithms (NEWBLER and MIRA2 assemblers) using a variety of assembly parameters. The 'best' (optimal) assembly can be visually assessed from a number of graphical print outs which examine the total number of nucleotides sequenced, the average length of the contigs, the number of reads per contig and the number of unincorporated reads or 'singletons'. In this work, the assemblies that gave the 'best' (deepest and most even) coverage of the target enzyme encoding genes and those which leave fewest orphaned singletons were used.

Identification. RACE-PCR and full length Sanger sequencing

Following bioinformatics analysis, sequences related to plant cell wall degrading enzyme cDNAs were retrieved from each 454-derived EST dataset via BlastX searches and also detailed visual examinations of potential enzyme active sites. Full-length cDNA sequences were obtained using classical molecular techniques such as 'Rapid amplification of cDNA ends' or RACE PCR. To exclude the possibility that candidate genes came from microorganisms associated with the beetle's midgut flora (which should have been largely washed away) or actually infecting the midgut tissue, specific primers from each cDNA sequence were designed and used in PCR experiments using, as a template, genomic DNA prepared from a tissue which is not the midgut (legs for example). The presence of a PCR product indicates that these genes truly belong to the insect genome and were not derived from a contaminating microorganism.

Figures 1-6 show amino acid sequence alignments of each class of enzyme, using known sequences for which the crystal structure has been resolved as a reference. In Figure 1, the reference sequence is the endoglucanase from Humicola insolens (Davies et al. (1995) Biochemistry vol. 34 pp 16210- 16220), not including the additional cellulose binding domain lacking in beetle enzymes; in Figure 2 it is the cellulose Cell48F from Clostridium cellulolyticum (Parsiegla et al. (2008) J. Mol. Biol. vol. 375 pp 499-510), not including the additional cellulose binding domain lacking in beetle enzymes; in Figure 3 it is the endopolygalacturonase II from Aspergillus niger (van Santen et al. (1999) J. Biol. Chem. vol. 274 pp 30474-30480); in Figure 4 it is the pectin methylesterase from Erwinia chrysanthemi (Fries et al. (2007) EMBO J. vol. 26 pp 3879-3887); in Figure 5 it is the β- mannanase from the blue mussel Mytilus edulis (Larsson et al. (2006) J. Mol. Biol. vol. 357 pp 1500-1510); and in Figure 6 it is the RhiE protein, a rhamnose-induced protein from the plant bacterial pathogen Erwinia chrysanthemi (Laatu & Condemine (2003) J. Bacteriol. vol. 185 pp 1642-1649). In each case, to facilitate the alignment, the predicted amino -terminal signal peptide of each beetle enzyme (included in SEQ ID NOs: 1-71 and 143-147) has been removed. These figures show the sequence conservation of the enzymes between different types of beetle.

Functional expression and enzyme characterisation

Following the RACE-PCR or full length candidate genes, all of the genes are cloned into shuttle vectors (such as pCR4-TOPO/TA) in E. coli. Such E. coli vectors facilitate the transfer of beetle cDNAs to either yeast or into insect cells. Enzymatic activities associated with lysates of E. coli are analysed. The beetle enzymes will achieve the correct post-translational processing in insect cells or yeast, therefore giving a more accurate picture of their catalytic activities and efficiencies. For expression in the insect derived cell line Sf9, the pIB TOPO/TA expression vector (Invitrogen) is used. This vector adds a specific epitope tag (V5) at the carboxyl-terminus of each protein. Successful expression of each recombinant protein is assessed by Western blot using a specific antibody directed against the V5 epitope, thus avoiding the generation of specific antibodies for each recombinant protein. The predicted protein primary structures corresponding to two endo- -l ,4-glucanases, two cellulose l,4- -cellobiosidases and nine endopolygalacturonases cloned from C. tremulae midguts, show the presence of an amino -terminal signal peptide. This indicates that these proteins are secreted in the extra-cellular space by midgut cells. The recombinant proteins expressed in Sf9 cells are also secreted into the culture medium. The presence or absence of enzymatic activity is assessed for each recombinant protein by zymogram analysis, i.e., direct visualisation of the enzymatic activity after protein electrophoresis performed under non-reducing conditions, as well as by classical enzymatic activity assays. Specific substrates are used, e.g., carboxymethyl-cellulose (CMC) as well as microcrystalline cellulose (MCC) for cellulases (endo- -l,4-glucanases and cellulose l,4- -cellobiosidase), pectic acid for endopolygalacturonases, esterified pectin for pectin methylesterases and mannan for endo-1 ,4- -mannosidases.

R Ai-facilitated control of insect growth

Double stranded RNA products encoding plant cell wall degrading enzymes and portions of these can be introduced into insects by (a) direct injection of dsR A into the insect hemocoel (open blood system); (b) feeding of the insects on the dsRNA by direct application to their food or via expression of dsRNA constructs in plants; and/or (c) expression of the constructs within the genome of the insect via germline mediated transformation, to provide proof of principle for knock-down of enzyme expression by R Ai.

Sections of the genes encoding for the polysaccharide degrading enzymes can be expressed in plants in constructs such as those disclosed in Li et al. (Plant Cell Rep. (2010) vol. 29 pp 113-123). Expression can be carried out in the tissues where the pest of interest is typically situated, e.g., roots for rootworm, or in given organelles if necessary, e.g., plant chloroplasts. Insects are weighed at different times after exposure to plants with and without RNAi constructs, or with and without direct exposure to dsRNA. Reduction in weight is recorded relative to the control. A repeatable reduction results in a reduction in the effective feeding damage which can be caused by the pest, or arrests development of the pest and/or its ability to reproduce (or reduces the numbers of its offspring). This is, therefore, a control/amelioration strategy for pest damage.

Codon usage in beetle species

A meta-analysis of all the publicly available coleopteran EST datasets present in the dbEST database at NCBI was performed. The codon usage of the 167 plant cell wall degrading enzyme messages identified was compared to the one from the overall ESTdatasets of each of the beetle species surveyed, as well as selected organisms such as Nosema bombycis, Wolbachia and Saccharomyces cerevisiae. The results are shown in Tables 3 and 4, with Table 3 showing the usage as a fraction of the total and Table 4 showing the usage according to relative synonymous codon usage (RSCU) values. RSCU values lower than 1 indicate that a codon is avoided and values higher than 1 indicate the given codon is preferred. A higher RSCU value indicates a higher preference for the particular codon. In both Tables, the preferred codon(s) for each amino acid is highlighted in italics. Table 2: Description of enzymes according to invention

Table 2: Description of enzymes according to invention

'Enzyme nomenclature and classification commission, http://www.chem.qmul.ac.uk/iubmb/enzyme/

bCarbohydrate-active enzyme database, http://www.cazy.org/; GH: glycoside hydrolase; CE: carbohydrate esterase; PL: polysaccharide lyase

¹ 1 got this information from the Cazy website. Please let me know if there are any problems with this.

2 Please can you provide some uses?

Table 3 - codon usage in plant cell wall degrading enzyme messages

#Codon (amino acid) PCWDEs C.maculatus C.tremulae G.viridula L.decemlineata S.oryzae T.castaneum D.melanogaster Nosema S.cerevisiae Wolbachia

GCA (A) 0,287 0,346 0,352 0,361 0,348 0,322 0,329 0,314 0,27 0,29 0,387

GCC (A) 0,236 0,201 0,219 0,204 0,211 0,215 0,2 0,234 0,22 0,22 0,146

GCG (A) 0,161 0,132 0,116 0,128 0,127 0,145 0,197 0,187 0,15 0,11 0,11

GCT (A) 0,316 0,321 0,312 0,306 0,314 0,318 0,274 0,265 0.36 0.38 0,357

TGC (C) 0,414 0,392 0,38 0,362 0,376 0,351 0,361 0,483 0,26 0,37 0,503

TGT (C) 0.586 0.608 0.62 0.638 0.624 0.649 0.639 0.517 0.74 0.63 0,497

GAC (D) 0,433 0,381 0,346 0,347 0,366 0,373 0,407 0,416 0,26 0,35 0,273

GAT (D) 0,567 0,619 0,654 0,653 0,634 0,627 0,593 0,584 0,74 0,65 0,727

GAA (E) 0,65 0,66 0,68 0,682 0,678 0,689 0,698 0,611 0,72 0,7 0,668

GAG (E) 0,35 0,34 0,32 0,318 0,322 0,311 0,302 0,389 0,28 0,3 0,332

TTC (F) 0,477 0,358 0,386 0,41 0,387 0,315 0,251 0,327 0,26 0,41 0,316

TTT (F) 0,523 0,642 0,614 0,59 0,613 0,685 0,749 0,673 0,74 0,59 0,684

GGA (G) 0,376 0,337 0,356 0,364 0,359 0,341 0,305 0,286 0,4 0,22 0,33

GGC (G) 0,198 0,203 0,185 0,185 0,184 0,197 0,213 0,273 0,12 0,19 0,201

GGG (G) 0,178 0,167 0,186 0,169 0,177 0,17 0,192 0,214 0,22 0,12 0,145

GGT (G) 0,248 0,292 0,274 0,282 0,28 0,292 0,29 0,227 0,17 0.47 0,324

CAC (H) 0,44 0,387 0,372 0,366 0,378 0,377 0,43 0,443 0,31 0,36 0,366

CAT (H) 0,56 0,613 0,628 0,634 0,622 0,623 0,57 0,557 0,69 0,64 0,634

ATA (1) 0,312 0,358 0,339 0,355 0,335 0,366 0,314 0,337 0,33 0,27 0,377

ATC (1) 0,315 0,244 0,24 0,24 0,241 0,207 0,171 0,222 0,16 0,26 0,213

ATT (1) 0,373 0,398 0,421 0,405 0,424 0,427 0,515 0,441 0,51 0,46 0,41

AAA (K) 0,612 0,665 0,674 0,676 0,683 0,712 0,963 0,819 0,69 0,58 0,667

AAG (K) 0,388 0,335 0,326 0,324 0,317 0,288 0,037 0,181 0,31 0,42 0,333

CTA (L) 0,134 0,13 0,12 0,127 0,115 0,127 0,107 0,104 0,11 0,14 0,136

CTC (L) 0,14 0,107 0,117 0,12 0,121 0,093 0,086 0,124 0,06 0,06 0,095

CTG (L) 0,202 0,15 0,146 0,148 0,147 0,125 0,094 0,151 0,07 0,11 0,11

CTT (L) 0,158 0,187 0,184 0,179 0,19 0,182 0,165 0,175 0,21 0,13 0,205

Table 3 - codon usage in plant cell wall degrading enzyme messages

TTA (L) 0,161 0,207 0,195 0,188 0,196 0,27 0,325 0,223 0.38 0.28 0,259

TTG (L) 0,206 0,219 0,238 0.239 0,231 0,203 0,222 0.223 0,17 0.29 0,195

ATG (M) 1 1 1 1 1 1 1 1 1 1 1

AAC (N) 0,435 0,398 0,362 0,356 0,382 0,367 0,334 0,373 0,27 0,41 0,338

AAT (N) 0,565 0,602 0,638 0,644 0,618 0,633 0,666 0,627 0,73 0,59 0,662

CCA (P) 0,398 0,389 0,405 0,401 0,39 0,379 0,345 0,354 0,33 0,42 0,398

CCC (P) 0,188 0,167 0,186 0,167 0,178 0,171 0,191 0,213 0,13 0,15 0,152

CCG (P) 0,16 0,13 0,117 0,131 0,135 0,15 0,207 0,202 0,16 0,12 0,104

CCT (P) 0,255 0,314 0,291 0,301 0,297 0,3 0,257 0,231 0,38 0,31 0,346

CAA (Q) 0,629 0,591 0,616 0,619 0,615 0,62 0,702 0,598 0,69 0,69 0,644

CAG (Q) 0,371 0,409 0,384 0,381 0,385 0,38 0,298 0,402 0,31 0,31 0,356

AGA (R) 0,252 0,342 0,372 0,359 0,346 0,339 0,261 0,225 0,48 0,48 0,406

AGG (R) 0,219 0,208 0,211 0,19 0,198 0,186 0,144 0,16 0,24 0,21 0,211

CGA (R) 0,167 0,146 0,151 0,175 0,165 0,158 0,192 0,182 0,1 0,07 0,104

CGC (R) 0,087 0,087 0,071 0,073 0,073 0,084 0,117 0,151 0,04 0,06 0,093

CGG (R) 0,134 0,084 0,086 0,081 0,09 0,093 0,115 0,14 0,06 0,04 0,066

CGT (R) 0,141 0,134 0,109 0,122 0,128 0,14 0,17 0,142 0,08 0,14 0,12

AGC (S) 0,147 0,14 0,12 0,108 0,114 0,123 0,119 0,169 0,07 0,11 0,168

AGT (S) 0,17 0,185 0,177 0,181 0,178 0,193 0,2 0,171 0,25 0,16 0,195

TCA (S) 0,25 0,228 0,253 0,261 0,243 0,21 0,225 0,187 0,21 0,21 0,221

TCC (S) 0,15 0,139 0,146 0,133 0,149 0,142 0,125 0,156 0,08 0,16 0,121

TCG (S) 0,111 0,094 0,09 0,107 0,102 0,106 0,14 0,143 0,09 0,1 0,06

TCT (S) 0,172 0,214 0,214 0,211 0,214 0,226 0,191 0,174 0,3 0.26 0.235

ACA (T) 0,306 0,367 0,37 0,381 0,36 0,361 0,365 0,347 0,32 0,3 0,322

ACC (T) 0,222 0,2 0,212 0,184 0,198 0,192 0,17 0,199 0,14 0,22 0,204

ACG (T) 0,186 0,142 0,124 0,133 0,134 0,149 0,178 0,179 0,19 0,14 0,115

ACT (T) 0,287 0,291 0,294 0,302 0,308 0,298 0,287 0,275 0,35 0,35 0.359

Table 3 - codon usage in plant cell wall degrading enzyme messages

GTA (V) 0,236 0,273 0,255 0,269 0,254 0,28 0,245 0,225 0,33 0,21 0,317

GTC (V) 0,215 0,187 0,18 0,183 0,186 0,178 0,172 0,196 0,14 0,21 0,131

GTG (V) 0,23 0,213 0,224 0,222 0,205 0,186 0,205 0,257 0,18 0,19 0,201

GTT (V) 0,318 0,327 0,341 0,326 0,355 0,356 0,378 0,322 0,35 0,39 0,351

TGG (W) 1 1 1 1 1 1 1 1 1 1 1

TAC (Y) 0,464 0,378 0,341 0,346 0,356 0,348 0,348 0,351 0,38 0,44 0,335

TAT (Y) 0,536 0,622 0,659 0,654 0,644 0,652 0,652 0,649 0,62 0,56 0,665

TAA (*) 0,385 0,384 0,355 0,333 0,37 0,479 0,544 0,458 0,7 0.47 0,466

TAG (*) 0,193 0,243 0,221 0,226 0,216 0,226 0,179 0,212 0,1 0,23 0,241

TGA (*) 0,422 0,373 0.424 0.441 0.413 0,295 0,277 0,33 0,2 0,3 0,293

Table 4 - codon usage in plant cell wall degrading enzyme messages

GCA (A) 4 4 5 5 5 3 2 5 3 5 7

GCC (A) 2 2 1 1 1 1 1 3 2 3 1

GCG (A) 1 1 0 0 0 0 0 1 0 0 0

GCT (A) 4 4 3 3 3 3 2 4 7 10 7

TGC (C) 6 6 5 5 5 5 3 8 1 2 15

TGT (C) 17 17 18 20 19 19 13 17 112 8 7

GAC (D) 7 7 4 5 4 4 3 4 4 10 2

GAT (D) 19 19 18 19 iz 16 9 13 38 37 16

GAA (E) 24 24 25 26 25 22 14 18 55 45 20

GAG (E) 6 6 4 4 4 3 2 5 1 6 3

TTC (F) 9 9 12 12 12 7 4 6 4 9 7

TTT (F) 21 21 3S 36 40 46 43 37 40 26 45

GGA (G) 11 11 7 6 6 6 2 4 9 2 3

GGC (G) 2 2 1 1 1 1 1 4 0 1 1

GGG (G) 2 2 1 1 1 1 0 2 2 0 0

GGT (G) 3 3 3 3 3 3 2 2 4 11 3

CAC (H) 6 6 5 5 5 4 3 6 1 3 5

CAT (H) 1∑ 1∑ 18 20 18 16 10 16 12 13 IZ

ATA (1) 6 6 12 13 12 14 5 10 9 5 15

ATC (1) 6 6 4 4 4 3 1 3 2 5 4

ATT (1) 11 11 15 15 15 16 28 13 42 15 16

AAA (K) 28 28 41 3S 41 47 325 7 67 41 45

AAG (K) 9 9 6 6 6 6 0 3 10 15 11

CTA (L) 1 1 1 1 1 1 0 1 1 1 2

CTC (L) 1 1 1 1 1 0 0 1 0 0 1

CTG (L) 3 3 2 2 2 1 0 2 0 1 1

CTT (L) 1 1 3 3 3 3 2 3 5 1 5

Table 4 - codon usage in plant cell wall degrading enzyme messages

#Codon (amino acid) PCWDEs C.maculatus C.tremulae G.viridula L.decemlineata S.oryzae | T.castaneum | D.melanogaster 1 Nosema S.cerevisiae Wolbachia

TTA (L) 1 1 3 3 3 9 S 5 18 8 9 TTG (L) 3 3 5 5 5 5 4 5 2 9 4

ATG (M) 18 18 19 20 18 16 10 16 22 20 16

AAC (N) 10 10 8 8 9 9 7 8 5 12 8

AAT (N) 27 27 31 31 30 32 2S 27 43 35 33

CCA (P) 8 8 7 6 7 6 3 7 3 9 6

CCC (P) 1 1 1 1 1 1 0 2 0 1 0

CCG (P) 1 1 0 0 0 0 1 2 0 0 0

CCT (P) 3 3 3 3 3 3 1 2 6 4 5

CAA (Q) 27 27 24 23 23 21 16 21 18 13 22

CAG (Q) 8 8 7 7 1 6 2 7 2 2 6

AGA (R) 5 5 10 10 9 9 3 3 11 21 9

AGG (R) 3 3 2 2 2 2 0 1 2 4 2

CGA (R) 2 2 1 1 1 1 1 2 0 0 0

CGC (R) 0 0 0 0 0 0 0 1 0 0 0

CGG (R) 1 1 0 0 0 0 0 1 0 0 0

CGT (R) 1 1 0 0 1 1 1 1 0 2 0

Claims

1. A Coleoptera polysaccharide degrading enzyme comprising an amino sequence which is:

a) at least about 55% identical to SEQ ID NO:67;

b) at least about 45% identical to SEQ ID NO: 143;

c) at least about 40% identical to SEQ ID NO:55;

d) at least about 65% identical to SEQ ID NO: 1 ;

e) at least about 86% identical to SEQ ID NO: 16;

f) at least about 81 % identical to SEQ ID NO:26;

g) at least about 98% identical to SEQ ID NO:51 or 62; or

or an enzymatically functional fragment or variant of any of these.

2. An enzyme according to claim 1 which is at least about 55% identical to SEQ ID NO: 67 and one or more of SEQ ID NOs:68-71 and comprises at least one of SEQ ID NOs:158 and 159.

3. An enzyme according to claim 1 which is at least about 45% identical to SEQ ID NO: 143 and one or more of SEQ ID NOs:144-147 and comprises at least one of SEQ ID NOs: 153-157.

4. An enzyme according to claim 1 which is at least about 40% identical to SEQ ID NO:55 and one or more of SEQ ID NOs:56-61 and comprises at least one of SEQ ID NOs:160-162.

5. An enzyme according to claim 1 which is at least about 65% identical to SEQ ID NO: l and one or more of SEQ ID NOs:2-15 and does not comprise a cellulose binding domain.

6. An enzyme according to claim 1 which is at least about 86% identical to SEQ ID NO: 16 and one or more of SEQ ID NOs: 17-24 and comprises at least one of SEQ ID

NOs:163 and 164.

7. An enzyme according to claim 1 which is at least about 81% identical to SEQ ID NO:26 and one or more of SEQ ID NOs:27-50 or 52-54 and comprises SEQ ID NO: 165, or which is at least about 98% identical to SEQ ID NO:51 and comprises SEQ ID NO:165.

8. An enzyme according to claim 1 which is at least about 98% identical to SEQ ID NO:62 and one or more of SEQ ID NOs:63-66 and comprises at least one of SEQ ID NOs:166 and 167.

9. A method of degrading plant material comprising exposing the material to at least one polysaccharide degrading enzyme according to claim 1 -8.

10. A method according to any preceding claim comprising expressing the enzyme in a micro-organism.

1 1. A method according to claim 10 comprising contacting the plant material with the micro-organism and/or contacting the plant material with an extract of the microorganism and/or an extracellular growth medium of the micro-organism.

12. A method according to any of claims 9-11 wherein the amino acid sequence of the enzyme is encoded by any of polynucleotide sequences SEQ ID NOs:72-142 or 148- 152.

13. A micro-organism comprising at least one polysaccharide degrading enzyme according to any of claims 1 -8.

14. A micro-organism according to claim 13 comprising at least one of the polynucleotide sequences SEQ ID NOs:72-142 or 148-152 or a micro-organism equivalent thereof.

15. A micro-organism according to claim 13 or 14 which is a yeast or a bacterium.

16. A plant comprising at least one polysaccharide degrading enzyme according to any of claims 1 -8.

17. A plant according to claim 16 comprising at least one of the polynucleotide sequences SEQ ID NOs:72-142 and 148-152 and/or a portion thereof, or a plant equivalent of such a sequence or portion.

18. A polynucleotide encoding a polypeptide according to claim 1.

19. A polynucleotide according to claim 18 comprising at least one of SEQ ID NOs:72- 142 and 148-152.

20. An expression vector comprising at least one polynucleotide according to claim 18 or 19.

21. A method of controlling an insect pest comprising feeding to the insect a plant according to claim 16 or 17.

22. A method according to claim 21 wherein R A interference occurs in the insect.