CA2190194A1 - A xylanase obtained from an anaerobic fungus - Google Patents

Abstract

A xylanase gene, denoted xynC, encoding a novel xylanase (XynC) obtained from strains of the anaerobic fungus Neocallimastix patriciarum is provided. The DNA
sequence of the xynC gene is also provided. Transformation of microbial and plant hosts with the xynC gene is described. The xynC gene may be used to design probes for use in hybridization experiments to isolate xylanase genes from other anaerobic fungi. The xynC gene has been used to construct an oleosin-xynC expression construct encoding an oleosin-xylanase fusion protein which retains xylanase activity.
Transgenic Brassica napus (canola), transformed with the oleosin-xynC expressionconstruct, expresses the oleosin-xylanase fusion protein which is immobilized in the oil-body membrane of the B. napus seeds. Canola meal, the protein-rich residue left after canola oil is extracted from canola plants, when derived from the transgenic B. napus of the present invention, retains substantial xylanase activity, making it an ideal animal feed supplement.

Description

2~ 901 q4 A XYLANASE OBTAINED FROM AN ANAEROBIC FUNGUS

FIELD OF THE INVENTION
The present invention relates to the field of molecular biology. In particular, the invention relates to genes encoding xylanases obtained from strains of the anaerobic fungus Neocallimastixpatriciarum.

BACKGROUND OF THE INVENTION
Endo-xylanases are enzymes that randomly cleave the ,B(1-4) linkages between xylose residues making up the backbone of xylans, a prevalent form of hemicellulose found predominantly in plant primary and secondary cell walls. If this complex plant cell wall poly~accl1d~ide is hydrolyzed with xylanases, it can be exploited as a rich source of carbon and energy for the production of livestock and microo,yd"i:,",s.
Enzymatic disruption of plant cell walls also increases the efficiency of a number of industrial processes such as juice extraction, retting of flax fibres and pulp production.
As discussed in greater detail herein, it will be applt:cidL~:d that plant cell walls are highly variable structures containing several forms of hemicell~ se. Thus, the need exists to identify and produce novel xylanases that are efficient at degrading this complex polysaccharide.
The plant cell wall is a highly variable, complex and resilient structure encasing essentially every cell of higher plants. It ,~ st" ,l~ a rich store of carbon and energy for herbivores as well as an important renewable resource utilized by the pulp and paper, lumber, food, and phal" ,aceutical industries. The plant cell wall consists largely of polysac~il ,alicles and contains lesser amounts of lignin (phenolic esters) and protein.
The primary polysaccharide components of plant cell walls are cellulose (a hydrogen-bonded ,B(1-4)-linked D-glucan), hemicellulose, and pectin (McNeil et al., 1984). Fibrils of cellulose t:",bedded in a matrix of pectin, hemicellulose (comprising various ~-xylan polymers), phenolic esters and protein produce a protective structure resistant to dehydration and p~ lldLiol1 by phytopathogens through mechanical and enzymatic " ,echal1i~" ,:,.
Hemicellulose, the second most prevalent polysac-,l ,al ide in many plant cell walls is composed mainly of xyloglucan or xylan polymers. Xyloglucans consist of a ~ 21 qol 94 backbone of ~-4-linked-D-glucosyl residues sllhstitlltPd with a-linked D-xylosyl side chains, some of which are extended by fucose, galactose or arabinose residues (McNeil et al., 1984). Xylans have a backbone structure of ~(1-4)-linked xylose residues. The structure of xylan is co",, ' ~' by the dlldulll"~"l of various side chains (e.g., acetic 5 acid, arabinose, coumaric acid, fenulic acid, glucuronic acid, 4-~methylglucuronic acid) to the xylose residues (McNeil et al., 1984). The strands of hemicellulose are hydrogen bonded to cellulose fibrils to form a strong interconnected lattice.
Cell wall collll,Oailion varies with plant species, variety, tissue type, growthconditions, and age. DiKerences in cell wall composition have been reported between 10 dicotyledonous and monocotyledonouos plants (Chesson et al., 1995). The primary cell walls of all dicots and many monocots contain greater amounts of xyloglucan thanarabinoxylan. In contrast, plants belonging to the family Gramineae (e.g., grasses and cereal) have primary walls in which only cellulose is more abundant than arabinoxylan.
Higher pectin cu"c~ ldliOlls are found in the exterior wall or middle lamellae than in the 15 primary or secondary cells walls. Finally, as cells age, cell walls may become more lignified and resistant to microbial attack.
The complexity of the plant cell wall is related not only to cu" ,~.. ,al variation but also to the high degree of interaction between constituent cellulose, hemicellulose and pectin molecules. Dual i"lt:l",e~l,i"g networks of polysaccharides, comprising 20 cellulose fibrils crosslinked with hemicellulose and pectic polysacchraides linked by calcium bridges, not only produce a resilient primary cell wall but are of direct relevance to enzymatic dey,dddliol1 (Chesson et al., 1995).
Digestion of the plant cell wall is further cu",, I -' by the structure of polysau~,ha,ides. Cellulose is a simple unsubstituted polymer of ~(1-4)-linked glucose 25 and requires an endoglucanase and cellobiase for complete degradation. In cu",~-ali:,on, highly sl Ih~t:~ If''d arabinoxylan requires up to seven different enzymes for complete degradation. An endo-xylanase randomly cleaves the xylan backbone into xyloc'~, ',arides which are 5llh~Pt~ ntly degraded to xylose by a xylosidase.
Substituents are cleaved from the xylan backbone with arabinofuranidase, acetylxylan 30 esterase and a-glucuronidase. Ferulic and ~coumaric acid crosslinks are degraded by feruloyl and ~coumaryl esterases. If complete degradation of the arabinoxylan is not required, fewer enzymes may be needed I iquef~ tirn of arabinoxylan requires ~ 2~ 9Cl q4 only the ~I,oll~l,i"g of the xylan polymers. Consequently, this objective may beachieved by the production o~ XYIOC' Jnl ' ,arides through the action of a single endo-xylanase. The choice of enzymes is dependent upon the substrate to be degraded.
The known a,, ' ~s of xylanases are numerous. For instance, the treatment 5 of forages with xylanases (along with cellulases) to increase the rate of acid production thus ensuring better quality silage and improvement in the subsequent rate of plant cell wall digestion by numinanst has been described. Xylanases can be used to treat rye, and other cereals with a high arabinoxylan content to improve the digestibility of cereal by poultry and swine. Xylanases can be used in bioconversion involving the hydrolysis~0 of xylan to xyi ' ~ hal i.les and xylose which may serve as growth substrates for uorydni~llls. This could involve simultaneous saccharification and ~ullt:llldliom Xylanases can be used in biopulping to treat cellulose pulps to remove xylan impurities to produce pulps with different characteristics. In some cases they can be applied to reduce the amount of chlorine needed to bleach the pulp and reduce the energy of15 refining pulp. Further, xylanases are useful in the retting of flax fibres, the clarification of fruit juices, the plt~,ua~dtion of dextrans for use as food thickeners and the production of fluids and juices from plant materials.
Some cha, d~ , of an endo-xylanase from N. patriciarum strain 27 (from the Agriculture and Agri-Food Canada Lethbridge culture collection) have been reported 20 previously (Tamblyn Lee et al., 1993). Tamblyn Lee et al. described the isolation of a 6.~-EcoRI fragment containing a gene encoding an endo-xylanase. The N. patriciarum strain 27 was not disclosed or made publicly available. The location of the xylanase gene was narrowed down to a 3.6-kb EcoRI Sall fragment. Expression of the endo-xylanase gene in E. coli produced at least three proteins (51, ~8 and 68 kDa) 25 having xylanase activity. This study did not fully ~;l ,al dl,l~ the N. patriciarum strain 27 endo-xylanase gene. No attempt was made to detemmine the nucleotide sequence of the gene. Nucleotide sequence data is required to create an efficient fusion construct between the endo-xylanase gene and the sequences of a heterologous t~x,ui~ssiun system. Without this i"~ul" IdliUI 1, the large DNA fragments of Tamblyn Lee 30 et al. would not be useful for the construction of a functional gene fusion. This effort would be hampered by a lack of detailed infommation about the stnucture of the gene and the location of useful restriction sites. The large DNA fragments identified by Tamblyn Lee et al. are not useful for~om~merlial enzyme production. Specifically, if these large DNA fragments were cloned into efficient uX~ 5~iull systems, translation of the resulting Lldl ,s.;,i,ul~ transcribed from a strong heterologous promoter would not be possible as translation would be 1~l " ,in dl~d at one of the multiple stop codons found in AT rich sequences upstream from the endo-xylanase gene. Further ~il ,aldul~ dlion, isolation and nucleotide sequencing of the N. patriciaNm strain 27 endo-xylanase gene would be required if it were to be of col"",el~ial importance.
In light of the many industrial a; ,:' -us for xylanases, the need for new xylanases is apparent. Accordingly, it would be of great importance to obtain genes encoding xylan-degrading enzymes from novel sources which may be brought to e,.~ Iu55iOI1 in other, high-producing microbial or eukaryotic ex~,ussiol1 systems.

SUMMARY OF THE INVENTION
In ac~;ou~dnce with the present invention, DNA sequences encoding novel and useful xylanases derived from anaerobic fungi are provided. As used herein and in the claims, the term "xylanase" means an enzyme having xylan degrading activity.
A xylanase gene (xynC) from Neoc~"U~a~ patriciaNm strain 27 from the Agriculture and Agri-Food Canada culture collection at Lethbridge, Alberta, Canada has been cloned and sequenced, and the nucleotide sequence of a DNA fragment including xylanase encoding region (CDS) of the xynC gene is provided in SEQ ID NO. 1.
Escherichia coli strain DH5a (pNspX-04), canying the xynC gene was deposited November 8, 1996 with the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland, 20852-1776, as ATCC98249) .
The invention extends to DNA sequences which encode xylanases and which are capable of hybridizing under stringent conditions with all or part of the xynC gene sequence. As used herein and in the claims, "capable of hybridizing under stringent condilio,1~" means annealing to a subject nucleotide sequence, or its complementary strand, under standard conditions (ie. high temperature and/or low salt content) which tend to disfavor annealing of unrelated sequences. As used herein and in the claims, ~conditions of low stringency" means hyulidi~dliol1 and wash conditions of 40 - 50~C, 6 X SSC and 0.1 % SDS (indicating about 50 - 80% homology). As used herein and in the claims, "~iu~ Idiliul~s of medium stringency~ means hybl idi~dlion and wash conditions 219~194 of 50 - 65~C, 1 X SSC and 0.1% SDS (indicating about 80 - 95% homology). As usedherein and in the claims, "conditions of high stringency" means hybridi~dlioll and wash conditions of 65 - 68~C, 0.1 X SSC and 0.1% SDS (indicating about 95-100%
homology) .
A method for identifying other nucleic acids having xylanase activity is also provided wherein nucleic acid molecules are isolated from an organism and nucleic acid hybl idi~dliu n is perfommed with the nucleic acid molecules and a labelled probe having a nucleotide sequence that includes all or part of nucleotide sequence SEQ ID NO. 1.
By this method, xylanase genes similar to the xynC gene may be identified and isolated from other anaerobic fungi.
The invention extends to purified and isolated xylanases obtained from strains of Neocr"'"a:,li,~ patriciaNm, particularly Neor~"'"a:,li,~ patriciaNm strain 27. A
preferred xylanase has the amino acid sequence shown in SEQ ID NO. 2.
The invention extends to ~ iol l constructs constituting a DNA having a coding region encoding a xylanase of the present invention operably linked to control sequences capable of directing ~ ,sion of the xylanase in a suitable host cell. The control sequences may be homologous to or h~l~lulogous to the xylanase encoding region. As used herein and in the claims, the temm "homologous" DNA refers to DNA
originating from the same species as the host cell or control sequences, as the context requires. For example, Aspergillus nigermay be transformed with DNA from A. niger to improve existing properties without introducing properties that did not exist previously in the species. As used herein and in the claims, Lheterologous" DNA refers to DNA
originating from a diflerent species. For example, the N. patriciaNm strain 27 xynC may be cloned and e,~ ssed in E. coli.
The invention further extends to host cells which have been lldn~fulllled with, and express DNA encoding a xyianase of the present invention, and to methods of producing such transformed host cells. As used herein and in the claims, "host cell"
includes animal, plant, yeast, fungal, protozoan and prokar,votic host cells.
The invention further extends to lldnsy~l ,iu plants which have been transfommedwith a DNA encoding a xylanase of the present invention so that the transfommed plant is capable of ~ ssi"g the xylanase and to methods of producing such transfommed plants. As used herein and in the claims, "Lldllsg~,lic plant" includes lldnsge,,ic plants, 2~ 9~ 7~

plant tissues and plant cells. In a prefenred t", Ibodi, "~"1, the transformed plant is of the species Brassica napus (canola).
The present invention also extends to oleosin-xylanase fusion proteins, DNA
sequences encoding oleosin-xylanase fusion proteins, and 11 dl ,sge"ic plants, preferably S B. napus, which have been llan:~lurllled to express such oleosin-xylanase fusion proteins. Surprisingly, these oleosin-fusion proteins have been discovered to retain xylanase activity. When B. napus is transformed with a DNA sequence of the present invention encoding an oleosin-fusion protein, the oleosin-fusion protein may i"""~ Hn the IlltnllL)ldlle sunrounding the oil-bodies found in the B. napusseeds.
Iû The canola oil is extracted from the seeds by, for instance, crushing, leaving a solid fraction and an oil fraction. Disruption of oil-body membranes in the oil fraction leaves the oil-body ",e",l,,dnes forming a gum which can be separated from the oil. The gum contains the oleosin-xylanase fusion protein. The gum is then added to the soildfraction during production of canola meal. Canola meal is a low-cost animal feed15 supplement which is high in protein. Canola meal made from lldllSg~lliC B. napus l,dn~ ",ed with a DNA sequence encoding an oleosin-xylanase fusion protein therefore also provides an excellent source of su~ " ,e, lldl xylanase for the animal.
Xylanases of the present invention are useful in a wide variety of a;, ':c " :lsinvolving the degradation of xylan. Accordingly, the invention extends to feed 20 su~ ",~"l:, containing a xylanase of the present invention. Such feed supplements may also contain other enzymes, such as, proteases, ~:PI~ CPC, phytases and acidpho~ dldses. The xylanase may be added directly to an untreated, pelletized, or otherwise pluce~ ed feedstuff, or it may be provided separately from the feedstuff in, for instance, a mineral block, a pill, a gel fommulation, a liquid fommulation, or in drinking 25 water.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts a constnuction pathway of plasmid constructs carrying an endo-xylanase gene cloned from N. patriciaNm 27.
Figure 2 is a schematic ,~p,~se"ldlion of the deletion analysis in which the 30 location of the endo-xylanase gene cloned from N. patriciarum 27 was determined. E
= EcoRI; H = Hindlll; P = Pvull; B = Bglll.

2t 9~1 ~4 Figure 3 is the nucleotide sequence of a fragment containing the endo-xylanase gene (xynC) cloned from N. patriciarum 27. The predicted amino acid sequence is shown beneath the nucleotide sequence. The CDS is located from nucleotide 301 tonucleotide 1755.
Figure 4 is a schematic ~ sel lldliun of: A) the structure of the xynC coding region. The signal sequence is followed by the sequence encoding for the catalytic domain of the enzyme (solid box) and a repeated peptide (shaded box). The 3' end of the gene codes for a region of unknown function; and B) the oleosin-xylanase C
t9Xp~5~iul I constnuct. The endo-xylanase gene was ligated between the oleosin gene (promoter plus coding region) and the temminator of nopaline synthetase (NOS).
Figure 5 is a schematic representing the N. patriciarum endo-xylanase gene (xynC) fragments cloned into pGEX-4T-3. (Pvu - Pvull) DETAILED DESCR~r~llON OFTHE INVENTION
The present invention provides purified and isolated DNA sequences of anaerobic fungal origin, which encode xylanases and genetic variants thereof. The DNA sequence preferably includes the xylanase-encoding region (CDS, protein coding sequence). Genetic variants include hybrid DNA sequences containing the xylanaseCDS fused to regulatory regions such as promoter, leader peptide and terminator signals, originating from hollloloyuus or h~l~lulo!Juus sources. Genetic variants also include DNA sequences encoding mutant xylanase proteins and degenerate DNA
sequences wherein the xylan-degrading activity of the enzyme is retained. The present invention provides the starting material for the construction of "second generation'' xylanases, i.e., mutant xylanases with properties that difler from those of the enzymes isolated herein, or DNA sequences (encoding the xylanase CDS) altered to reflect the degeneracy of the genetic code or cross-species variation. Genes can be readily mutated by procedures known in the art (e.g., chemical, site directed, random polymerase chain reaction mutagenesis~ thereby creating gene products with altered properties (e.g., temperature or pH optima, specific activity or substrate specificity). The xylanase gene of the present invention can be used also in htlleluloyous hyl~lidi~dlion and polymerase chain reaction ~ Jel i" ,t:"l~, directed to isolation of xylanase-encoding genes from other natural sources.

Screening o,yani:,",s for endo-xylanase activity may be acomplished by a number of assays methods not critical to the present invention. These include visual assays such as the incorporation of xylan (e.g., oat spelt xylan, rye arabinoxylan) or .,hlulllogullic substrates (e.g., remazoi brilliant blue xylan or RBB-xylan) into agar media. Hydrolysis of the xylan will be indicated by the presence of zones of clearing around isolates with endo-xylanase activity. Staining of the medium with Congo red (Teather and Wood,1982) allows visualization of the hydrolytic activity on solid medium containing non-"l " u" ,ogt:",c substrates such as oat spelt xylan.
Once a xylanase of interest has been identified, the DNA sequence encoding such a xylanase may be cloned from the organism which naturally produces the xylanase by a variety of methods. Gene libraries (genomic DNA and/or cDNA) are constructed by standard methods (Ausubel et al., 1990; Sambrook et al., 1989) and screened for the desired gene. In the case of eukaryotic olyd"i~",s and inducible xylanase exp,t,s:,iol1, it may be advantageous to construct cDNA libraries with mRNA
isolated from the organism, which naturally produces the xylanase, following cultivation in an inducing medium (e.g., a medium containing straw or xylan as the soie carbon source). Clones carrying the desired xylanase gene may be isolated by screening the library with enzyme activity assays (Teather and Woods, 1982), heterologous probes, or results generated during purification of the gene product, such as N-terminal and internal amino acid sequence data and a, ltiL,o.lies.
Using Congo red detection, a A'~ r " lldstixpatricianum strain 27 genomic DNA
library was screened for lambda clones possessing xylanase activity (Tamblyn Lee et al.,1993). A xylanase positive clone carrying a 6.5-kb EcoRI insert was identified and confinmed by Southem blot hybridization to have originated from N. patriciarum strain 27.
Plasmid DNA extracted from the newly isolated clone and introduced into E. coli cells by t~dl1~ul'''dlion produced ampicillin resistant, xylanase positive colonies.
Zymogram analysis of cell extracts from E. coR DH5a cells canying a 3.5-kb EcoRI Sall DNA fragment isolated from the original 6.5-kb EcoRI fragment showed active bands of 68, 58, and 51 kDa. The gene (xynC), encoding the observed xylanase activity in r~combillalll E. coli clones, was identified by deletion and nucleotide sequence 2t 901 94 analysis. The nucleotide sequence and deduced amino acid sequence are shown in Figure 3, and further illustrate that the cloned sequence encoded a xylanase.
It is known in the biological arts that certain amino acid ~llhstitl~tiQns can be made in protein sequences without affecting the function of the protein. Generally, 5 conservative amino acid sl Ihstitl ~tions are tolerated without affecting protein function.
Similar amino acids can be those that are similar in size and/or charge properties, for example, aspartate and glutamate and isoleucine and valine are both pairs of similar amino acids. Similarity between amino acid pairs has been assessed in the art in a number of ways. For example, Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure, Volume 5, Supplement 3, Chapter 22, pages 345-352, which is i"col~.olaled by reference herein, provides frequency tables for amino acid sl Ih~tih Itions which can be employed as a measure of amino acid similarity. Dayhoff et al.'s frequency tables are based on comparisons of amino acid sequences for proteins having the same function from a variety of evolutionarily different sources.
It is also known that often less than a full length protein has the function of the complete protein, for example, a truncated protein lacking an N-terminal, intemal or a C-terminal portion of the protein often has the biological and/or enzymatic activity of the complete natural protein. Those of ordinary skill in the art know how to make truncated protein and proteins with intemal deletions. In the present invention, the function of a 20 truncated xylanase protein or an intemally deleted xylanase protein can be readily tested using the xylanase assay described ht:l~il Ibelu.. and in view of what is generally known in the art.
Sl~hstitllt~d and truncated xylanase derivatives which retain subbldlllially thesame the enzymatic activity of the xylanase ~,c " 'Iy disclosed herein are con:,ide, ~d equivalents of the t~ ",, ' 'icd xylanase and are within the scope of the present invention, particularly where the specific activity of the c, Il- It~d or truncated xylanase derivative is at least about 10% of the specifically ~x~",, ' 'icd xylanase. The skilled artisan can readily measure the activity of a tnuncated or .sl Ih~tih It~d xylanase using the assay procedures taught herein and in view of what is generally known in the art.
This invention includes structurally variant xylanases derived from a xylanase obtained from an anaerobic fungi, particularly those derived from a xylanase ~e~ ic..ll"
disclosed herein, that are suL,:,Ldllli~lly functionally equivalent to that xylanase as ~1 901 74 assayed as described herein in view of what is generally known in the art. Structurally variant, functional equivalents of the xylanases of this invention include those xylanases of an anaerobic fungi having a contiguous amino acid sequence as in the xylanaseamino acid sequence disclosed herein (SEQ ID NO. 2), particularly those variant 5 xylanases which have a contiguous amino acid sequence of a xylanase of an anaerobic fungi that is a contiguous sequence at least about 25 amino acids in length.
As with other genes, it is possible to use the characterized xylanase-coding sequences from anaerobic fungi in a variety of ~X,ul'~55iOI1 systems for co"""ell.idl protein production. Application of ,~col"bi"al,l DNA I~:u-l",ology has enabled enzyme 10 manufacturers to increase the volume and efficiency of enzyme production, and to create new products. The original source organism need no longer limit the production of co"""e,uial enzymes. Genes encoding superior enzymes can be lldl1~ u~d from organisms such as anaerobic fungi, typically i" ,,ul dUliCdl for commercial production, into well .;I,ald.;l~ d industrial microbial production hosts (e.g., Aspergillus, Pichia, 15 Trichoderma, Bacillus spp.). As well, these genes may be transferred to novel plant and animal ~ iu" systems.
Industrial strains of ",i.;,uo~yani:,ll,s (e.g., Aspergillus niger, Aspergillus ficcum, Aspergillus awamori, Aspergillus oryzae, Tri,,l,od~""d reesei, Mucor miehei, Kluyveromyces lactls, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coll, 20 Bacillus subtilis or Bacillus li,,l,enlru""i~) or plant hosts (e.g., canola, soybean, corn, potato) may be used to produce xylanases. All systems employ a similar approach to gene exp,u:,:,iu". An t~AIn~ssioll construct is assembled to include the protein coding sequence of interest and control sequences such as promoters, enl1al,ce,:, and ~ l lil ldIul :~. Other sequences such a signal peptide sequences and selectable markers 25 may be included. To achieve extracellular ~,UI~:~Sioll of xylanase, the ex~ ssiol1 construct of the present invention utilizes a secretory signal peptide sequence. The signal peptide sequence is not included on the ~-,ul~ssion construct if cytoplasmic expression is desired. Tldnsc,i,uIiol1dl tellllilldIul~ are included to ensure efficient transcription. Ancillary sequences enhancing ~pl~5~iU" or protein purification may 30 also be included in the e~ s~ion construct. The promoter, enhancer, signal peptide and terminator elements are functional in the host cell and provide for efficient expression and secretion of the xylanase.

The xylanase-coding sequences are obtained from anaerobic fungal sources sources. Various promoters (l~d~,,,i,uliollal initiation regulatory region) may be used according to the present invention. The selection of the appropriate promoter isd~,uel1~ld"l upon the proposed ~,~,ul~SSiO11 host. The promoter may be ho",ologous or S heterologous to the cloned protein coding sequence. Examples of heterologous promoters are the E. coli tac and trc promoters (Brosius et al., 1985), Bacillus subtilis sacB promoter and signal sequence (Wong, 1989), aox1 and aox2 from Pichia pastoris (Ellis et al., 1985), and oleosin seed specific promoter from Brassica napus (van Rooijen and Moloney, 1 995a). Promoter selection is also d~,ut,l ~dt"l upon the desired 10 efficiency and level of peptide or protein production. Inducible promoters such tac and aox1 are often employed in order to dldllldlically increase the level of protein~A,ul~s~ium OVel~,UI~;OI1 of proteins may be harmful to the host cells.
Consequently, host cell growth may be limited. The use of inducible promoter systems allows the host cells to be cultivated to acceptable densities prior to induction of gene 15 expression, thereby, facilitating higher yields of product. If the xylanase-coding sequence is to be integrated through a gene I~Jlac~ l ll (omega insertion) event into a target locus, then promoter selection may also be influenced by the degree of homology to the target locus promoter.
Various signal peptides may be used according to the present invention. A
20 signal peptide sequence which is ho",ologous to the xylanase-coding sequence to be expressed may be used. Alternatively, a signal peptide sequence which has been selected or designed for improved secretion in the ~,~,ul~ion host may also be used.
For example, B. subtilis sacB signal peptide for secretion in B. subtilis, the Saccharomyces cerevisiae a-mating factor or P. pastoris acid phosphatase phol signal 25 sequences for P. pastoris secretion. A signal peptide sequence with a high degree of homology to the target locus may be required if the xylanase-coding sequence is to be integrated through an omega insertion event. The signal peptide sequence may be joined directly through the sequence encoding the signal peptidase cleavage site to the xylanase-coding sequence, or through a short nucleotide bridge consisting of usually 30 fewer than ten codons.
Elements for ~"hal1ui,lg t"~,u~SSiO11 lldl1scli,uliol1 (promoter activity) and lldl1sldliu" have been identified for eukaryotic protein expression systems. For 2~ 901 ~4 example, the po~iliuni"g the Cauliflower Mosaic Virus (CaMV) promoter 1000 bp oneither side of a h~ uloyùus promoter may elevate transcriptional levels by 10 to 400 fold. The expression construct should also include the app,uplidLu translationalinitiation sequences. Mo-liricdlion of the tl~,ultlssiùll construct to include the Kozak 5 consensus sequence for proper l~dnsldliollal initiation may increase the levei of llallsldlioll by 10 fold.
Elements to enhance purification of the protein may also be included in the u.~p,us~iu" construct. The product of oleosin gene fusions is a hybrid protein containing the oleosin gene joined to the gene product of interest. The fusion protein retains the 10 lipophilic properties of oleosins and is illcoi,uoldlt:d in the oil body membranes (van Rooijen and Moloney, 1995a). Association with the oil bodies may be exploited tofacilitate the purification of the ,uco",ui"a"l oleosin fusion proteins (van Rooijen and Moloney, 1995a).
A selection marker is usually employed, which may be part of the ex,ulussio 15 construct or separate from the C:A,UI~S~ioll construct (e.g., carried by the ~X,u~ iOIl vector). The selection marker may be used as an alternative target locus for t:~,u~ssion construct illlUyldliOIl. Tldll:~ulllldliull of the host cells with the lucollluilldlll DNA
molecules of the invention is monitored through the use of selectable markers.
Examples of these are markers that confer resistance to antibiotics (e.g., bla confers 20 resistance to ampicillin for E. coli host cells, nptll confers kanamycin resistance to B.
napus cells) or that pemmit the host to grow on minimal medium (e.g., HIS4 enables P.
pastoris GS115 His cells to grow in the absence of histidine). Sel~ctAhle markers are usuually associated with lldllscli~uliullal and l~dnsldlional initiation and IUIIIIilldliOIl regulatory regions different from the tlh~ ssion construct in order to allow for25 independent t~x,u~s:~ioll of the marker. Where antibiotic resistance is employed, the conce"l~dliull of the antibiotic for selection will vary depending upon the antibiotic, generally ranging between 10 and 500 ,ug of the alllibiulic/lllL of medium.
The tl~,u~s:,iu~, constnuct is assembled by employing known recombinant DNA
techniques. Restriction enzyme digestion and ligation are the basic steps employed to 30 join two fragments of DNA. The ends of the DNA fragment may require Illodi~icdliull prior to ligation and this may be accu" ,,u li~hed by filling in overhangs, deleting terminal portions of the fragment(s) with nucleases (e.g., Exolll), site directed mutagenesis, and ~1 9~1 ~4 adding new base pairs by the Polymerase Chain Reaction (PCR). Polylinkers and adaptors may be employed to facilitate joining of select fragments. The ex,u,~ssioll construct is typically as~e" ~uled in stages employing rounds of restriction, ligation and lldl1~iulllldliunofE coli. Therearenumerouscloningvectorsavailableforconstnuction S of the t:X,~ ssiu" constnuct and the particular choice is not critical to this invention. The selection of cloning vector will be influenced by the gene transfer system selected for introduction of the ~,u~ussioll contruct into the host cell. At the end of each stage, the resulting construct may be analyzed by restriction, DNA sequence, hyblidi~dIiu,, and PCR analyses.
Thet,,~p,us~iu,,constructmaybel,d,,~ul,,,edintothehostasthecloningvector construct, either linear or circular, or may be nemoved from the cloning vector and used as is or introduced onto a delivery vector. The delivery vector facilitates the introduction and "..~;.,t~,ndnce of the expression construct in the selected host cell type. The ~cu~t:s~iu~ I constnuct is introduced into the host cells by employing any of a number of 15 genetransfersystems(e.g.,naturalco",,u~tu"ce,~l,u",ica'!ymediatedl~d~ ul",dliul"
protoplast lldll~rulllldlioll, eleuIIupo,dlioll, biolistic lldl1~iUIIIIdIiOI1, Ird~ .Iiul1, or conjugation) and is dependent upon the host cells and vector systems used.
For instance, the expression construct can be introduced into P. pastoris, cellsby protoplast I,dn:,~u", IdtiO n or t:le~.IIupol..tion. Electroporation of P. pastoris is easily 2û accol"ul;~l,ed and yields Ildll~ulllldIiull efficiencies co",,ualdble to spheroplast transformation. Pichia cells are washed with sterile water and resuspended in a low conductivity solution (e.g., 1 M sorbitol solution). A high voltage shock applied to the cell suspension creates transient pores in the cell membrane through which the I~dn~uilllillg DNA (e.g., e~ lussion construct) enters the cells. The expnession25 construct is stably "IdillIdil,ed by integration, through homologous It~collluilldIiull~ into the aoxl (alcohol oxidase) locus.
Al~ cly, an ~,~,ul~ iu" construct, comprising the sacB promoter and signal sequence operably linked to the protein coding sequence, is carried on a plasmid, pUB110, capable of autonomously replicating in B. subtilis cells. The resulting plasmid 30 construct is introduced into B. subtilis cells by lldn~UlllldliUII. Bacillus subtilis cells develop natural co, Il,UUlt:l ,ce when grown under nutrient poor conditions.

Host cells carrying the t:A,u,~s~iun construct (i.e., l,dll~lu~ ed cells) are identified through the use of the selectable marker carried by the ~dA,UI ~S::~iOIl construct or vector and the presence of the gene of interest confimmed by a variety of techniques including h~,bl-idi~dliol1~ PCR, and antibodies.
Trdrl~ullllad microbial cells may be grown by a variety of techniques including batch and continuous 1~" "~"laliOIl on solid or semi-solid media. Tldn~ilul " ,ad cells are propagated under conditions optimized for maximal product to cost ratios. Product yields may be d~ dl 11 " 'Iy increased through the manipulation of cultivation parameters such as temperture, pH, aeration and media composition. Careful manipulation andmonitoring of the growth conditions for recombinant hyper-~x~ i"g E. coli cells may result in culture biomass and protein yields of 150 g (wet weight) of cells/L and 5 g of insoluble protein/L, respectively. Low ~:ullc~lllldliolls of a protease inhibitor (e.g., phenylmethylsulfonyl fluoride or pepstatin) may be employed to reduce proteolysis of the over-expressed peptide or protein. Altematively, protease deficient host cells may be employed to reduce or eliminate deyldddliull of the desired protein.
Following ~""enldtion, the microbial cells may be removed from the medium through du~ d,,l processes such as centrifugation and filtration. If the desiredproduct is secreted, it can be extracted from the cell free nutrient medium. Altematively, the culture or cell free medium may be used directly or concel Illdl~d (e.g., ullld~illldliol1, dehydration, Iyu~ h ' 1) and used in an ~ 1 requiring xylanase activity. In the case of intracellular production, the cells may be harvested and used directly or ruptured (e.g., rl ,e.;l Idl ,ical forces, ultrasound, enzymes, chemicals, high pressure). The resulting Iysate may be used as in an a,),' " n requiring xylanase activity or subjected to further p, uces:~il ,g.
In a third example, Brassica napus celis are l~dn~r~,l",ed by Agrobacterium mediated l~dn~ul,,,atioll. The t~X~ ssioll construct is inserted onto an binary vector capable of replication in A. h""~rdciensand ", ' " 1 into plant cells. The resulting contruct is lldl~iulllled into A. tu",~ld,,iel7s cells carrying an attenuated Ti or ~helper "plasmid. When leaf disks are infected with the ,~cu,,,ui, ,anl A. t~ rdcie"~ cells, the ~x,ul~:~sioll construct is lldll~rt~ d into B. napus leaf cells by conjugaHI l ' :" " n of the binary veuLul..ex~ iu" construct. The ~A~ iOIl constnuct integrates at random into the plant cell genome.

2~ 9~1 94 After selection and screening, lldl1a~u~ ed plant cells can be regenerated into whole plants and varietal lines of Irdnsgel1ic plants developed and cultivated using known methods.
Xylanase may be extracted from harvested portions or whole plants by grinding, S homogenization, and/or chemical treatment. The use of seed specific lipophilicoleo~i"..g~"e fusions can facilitate purification by partitioning the oleosin fusion protein in the oil fraction of crushed canola seeds and away from the aqueous proteins (van Rooijen and Moloney, 1995a).
Expression of xylanases of the present invention in Brassica napus (canola) is useful, particularly as the enzyme will be expressed in every seed of the plant. Canola is an important agricultural crop due to its high oil content. There are many uses for canola oil, including such diverse a" I " ns as lubricating oils and oils for human consumption. The non-oil fraction remaining after the oil is extracted from canola seeds by techniques such as cnushing may be described as canola meal. Canola meal is typically used as an animal feed supplement due to its high protein content, which may be as high as 40-50~/0. Canola meal makes an ideal feed supplement as it is sub~ldnlially less expensive than alternatives such as soybean meal. Furthemmore, canola meal also contains higher conce, IlldliUI ,:j than soybean meal of nutrients such as carbohydrates.
The oil in the seeds of B. napus is found within oil-bodies surrounded by an oil-body rl 1~ ~ Ibldl 1~1 which functions to contain the oil. Oleosin proteins are located in the rllt:lllbldlle surrounding the oil body. Oleosins (oil-body proteins) are structural proteins found in the seeds of all higher plants investigated to date (monocots, dicots and g~"""o~,uel",s). They are highly liophilic with a unique secondary structure which permits their central core to be embedded in oil-bodies while the more hydrophilic N-and C-temmini reside on the cytoplasmic side. Their role appears to be primarily that of stabilizing triacylglyceride-containing oil-bodies as discrete organelles (van Roijen and Moloney, 1 995a). The hydrophilic N- and C-temmini of the oleosin protein may provide dlldUI Illlt~l 11 sites for forming fusions with other proteins.
In a preferred embodiment of the present invention, B. napus is 11 dl l~ UI 11 ,ed with an ~p~ iull construct containing a nucleotide sequence encoding a xylanase of the present invention llal1sldliu,,,~lly fused to a nucleotide sequence encoding an oleosin 21 901 q4 .

protein to provide seed oil body t!X,u~u~siol1 of the xylanase, as described in the examples which follow.
The oleosin-xylanase fusion protein is immobilized in the seed oil-body membrane and remains with the canola meal portion during oil extraction. As 5 d~ on~ lud in the examples which follow, the oleosin-xylanase fusion proteins retain xylanase activity. Canola meal produced from the transgenic B. napus of the present invention thus provides an ideal source of xylanase when the canola meal is used as a feed supplement (protein source) in animal diets. Su,upl~ll ,e, lldl xylanase in animal diets degrades cell wall components in the animal feed, resulting in increased feed 10 digestion and a reduction in pollution from animal wastes.
If necessary, various methods for purifying the xylanase, from microbial ~t~ullt:llldlion and plant extracts, may be employed. These include pl~-;i,uildliol1 (e.g., ammonium sulfate pl~ui,uildliun)~ ulllullldluyldplly (gel filtration, ion exchange, affinity liquid ~,hlullldluyldplly), ulll "" " 1, elt:ul,uphoresis, solvent-solvent extraction (e.g., 15 acetone precipitation), cc." ~bi~ IdliOlls thereof, or the like.
All or a portion of the microbial cultures and plants may be used directly in a;, ' " 15 requiring the action of a xylanase. Various formulations of the cnude or purified xylanase p,t,pal ~s may also be prepared. The xylanase can be stabilized through the additions of other proteins (e.g., gelatin, skim milk powder) and chemical 20 agents (e.g., glycerol, polyethylene glycol, reducing agents and aldehydes). Enzyme su~,u~ ,iulls can be cu"c~"l, dl~d (e.g., tangential flow ull, d~ill, dliu") or dried (spray and drum drying, Iyupll' " 1) and formulated as liquids, powders, granules and gels through known p,uce~es.
Formulations of the desired product may be used directly in A!,' " 15 25 requiring the action of a xylanase. Liquid cunce"l,dlu~, powders and granules may be added directly to reaction mixtures and ~ m ~ dliUI 1::~. The fommulated xylanase can be administered to animals in drinking water. It may be mixed also with, sprayed on or pelleted with other feed stuffs through known processes. Alternatively, the xylanase gene may be introduced into an animal, thereby el;. "i, Idlil 19 the need for the addition 30 of extraneous xylanase.
In another fommulation, the xylanase of the present invention may take the form of viable microbial feed inoculants. Cultures of ",i,_,uolyani:,",s expressing a xylanase 2t ~01 9~
.

gene such as N. patriciarum strain 27 or, ucu" ,L i"a"l " ,iu, uo, ydl li~>l l l5 expressing the xylanase CDS are grown to high Cul lC~ ldliOl)s in fenmentors and then harvested and cu"cu"lldlt:d by ct"~ 9~qtion. Food-grade whey and/or other cryop,ul~uld"l~ are then admixed with the cell concentrate. The resulting mixture is then cryogu,)ically frozen 5 and freeze dried to preserve xylanase activity by standard Iyopl ,- - 1 procedures.
The freeze-dried culture may be further p,ucessed to fomm finished product by such further steps as blending the culture with an inert carrier to adjust the strength of the product.
All or a portion of the microbial cultures and plants as produced by the presentlû invention may also be used in a variety of industrial plucesses requiring the action of a xylanase.
Examples of such - r r~ 5 are in the production of feed i"y, udit:"l:, and feed additives for livestock production the retting of flax fibres the cld~ of fruit juices the pl~pdldliol1 of dextrans for use as food Illiukul1elx and the production of fluids and 15 juices from plant materials. Xylanases can be used also in the bioconversion involving the hydrolysis of xylan to xyloc --- ,arides and xylose and biopulping to treat cellulose pulps to remove xylan impurities to produce pulps with different Chal dutul i~lics.

2û EXAMPLES
Example 1. Cloning an endo-xylanase gene from Neocallimastixpatriciarum N~,---"'"a:,lixpatriciarum strain 27 was cultivated anaerobically at 39~C in a modified semi-defined medium (Lowe et al. 1985) containing either Whatman No. 1 filter paper or 0.15% glucose as a carbon source. Cells were harvested by 25 centrifugation after 4 d growth resuspended in extraction buffer (25 mM Tris-HCI pH
8.0; 10 mM EDTA; 50 mM glucose) and stored at-70~C overnight. The preparation was thawed at room temperature and ho",oy~ni~ed until all cells were resuspended.
Sodium dodecyl sulfate (SDS} and diethylpylucdllJol1dl~ (DEPC} were added to a final col1celllldliol1 of 0.5~/O (w/v) and 25 mM respectively. The suspension was incubated 3û at 37~C for 1 h. Flutt:i,,ase K (0.1 mg/mL) was added and the mixture was incubated for 1 h at 55~C extracted twice with phenol and twice with phenol/ul ,lol U~UI " ,. The DNA

21 901 ~4 was precipitated with ethanol and the resulting DNA pellet resuspended in TE (10 mM
Tris, pH 8.0;1 mM EDTA) buffer.
Neocallimastix patriciarum strain 27 genomic DNA was partially digested by EcoRI. Agarose gel purified 4- to 7-kb EcoRI fragments were ligated (overnight, 4~C) S to EcoRI-cut and dt,lJho~,uholylated AgtWESAB amms at a molar ratio of 1:2. The ligated DNA was packaged with a A DNA in vitro packaging kit. The phage library was amplified on TN plates (10 g Bacto-tryptone, 5 g NaCI per litre containing 0.2% maltose and 10 mM MgCI2, pH 7.5) with Escherichia coli ED8654 as the host bacterium.
R~oll II,il ,anl phage were screened for xylanase activity by overlaying plaqueswith 0.7~/O (wlv) agarose co"ldi"i"g 0.1-0.25% (w/v) water soluble oat spelt xylan dissolved in 25 mM potassium phosphate buffer (pH 6.5). The plates were incubated at 39~C for 3 -18 h and stained with a 0.1~/O (w/v) aqueous solution of Congo red and destained with 1 M NaCI. Xylanase-producing plaques were surrounded by a yellow halo visible against the red background. Two positive clones were recovered after screening 50,000 plaques. Positive plaques were picked and resuspended in SM buffer (Sambrooket al., 1989). The plaques were purified three successive times by isolation from agar plates.
Examole 2. Chalduleli~dliQll of positive endo-xylanase clones Phage stocks and DNA were prepared according to methods described by Sambrook et al. (1989). Restriction analyses detenmined that the two positive clones canried an identical 6.5-kb EcoRI insert. This clone was desiy"dled ANspX-101. The location of the endo-xylanase gene on the 6.5-kb EcoRI fragment was narrowed down by subcloning fragments of the 6.5-kb EcoRI fragment onto a number of cloning vectors including pBR322 (Bethesda Research l ~ ie:, - BRL, M -llg~, ON), pUC18 (BRL) or pBluescriptllSK+(Stratagene Cloning Systems, La Jolla, CA). [~,,l,e"~l,ia coli HB101 andDH5a(BRL)wereusedasthehostbacteriaforthevariouscloningvectors.
Tldn:,~ul,,,ed cells were p,.,paydl~d at 37~C in Luria-Bertani (LB) medium (Sambrook et al., 1989). Ampicillin (100 ,ug/mL) was illco,,uo,dl~d into media used to culture plasmid-bearing E. coiistrains. Tldlla~UIIIIdlll colonies were cultivated on LB medium containing 0.1-0.28% oat spelt xylan. Xylanase activity was detected by staining plates with Congo red.

~7 ~01~4 The 6.5-kb EcoRI insert from ANspX-101 was subcloned into pBR322 yielding plasmid pNspX-01 (Figure 1). The 1.7- and 2.2-kb Hindlll fragments from pNspX-01were cloned in the correct orit~ alion into pBR322 to produce pNspX-02. The 4.3-kb BamHI EcoRI fragment from pNspX-02 was ligated with BamHI EcoRI digested pUC18.
S The resulting plasmid was desiylldl~d pNspX-04 (Figure 1). Deletion of the Sall fragment from pNspX-04 produced pNspX-06. Further truncation of the 3.6-kb EcoRISallfragmentcontainedonpNspX-06wasacco,,,l,1ic,l,edbysubcloningtheEcoRlPvull or EcoRI Hindlll fragments on to pBluescriptSKII+ (Stratagene Cloning Systems).
Xylanase activity was observed only for the clone carrying the EcoRI Pvull fragment thereby su,, ,g that the Hindlll site was located within the endo-xylanase gene (Figure 2).
The origin of the endo-xylanase gene was confirmed by Southem blot hybl idi~dliUIl using the 3.5-kb Clal fragment from pNspX-02, labelled with [a-~P]-dCTP, as a probe (Tamblyn Lee et al.,1993).
ExamPle3. Bk,ch~",;cdlul~ald~ liu:,oftheclonedNe- ""a~ patriciaNmendo-xvlanase The cloned endo-~-1,4-xylanase was secreted into the pel i~ld~ i.; space of hostEscherichia coli (pNspX-06) cells. The biochemical ullaldult~ ,s of the cloned enzyme were dt~ l"illed using cnude extracts containing periplasmic endo-xylanase released by osmotic shock (Table 1, Tamblyn Lee et al., 1993). Xylanase activity was d~le",li"ed by measuring the amount of reducing sugars released from substrates according to the method of Nelson-Somogyi (Somogyi, 1952). The N. patriciarum endo-xylanase hydrolyzed oat spelt xylan and birch wood xylan almost equally well, but exhibited very low activity on arabinoxylan. The pH and temperature optima for the periplasmic endo-xylanase activity were 6.2 and 40~C, I~ e-,tiicly, and the Km for oat spelt xylan hydrolysis was 0.89 mg/mL. SDS-PAGE followed by zymogram analysis showed active bands of 68, 58, and 51 kDa. The isoelectric point, d~lell"i,led by isoelectric focusing combined with zymogram analysis, was 3.6.

~19019~

Table 1. General biochemical properties of the N. patriciarum 27 xylanase Property Molecularweight (kDa) 68158/51 Isoelectric point 3.6 pH optimum 6.2 Temperature optimum 40~C
Substrate specificity Oat spelt xylan +++++
Birch wood xylan +++++
Rye flour arabinoxylan +/-Carboxymethylcellulose ++
Acid-swollen cellulose +/-Barley ~-glucan +/-Lichenan Km (mg oat spelt xylan/ml) 0.89 Example 4. Nuclçotide seauence and structural analyses of the Neocallimastix patriciarum endo-xylanase gene The 3.4-kb EcoRI - Bglll fragment of pNspX-06 was sequenced in both strands.
Samples were prepared for DNA sequence analysis on an Applied Biosystems Model 373A DNA sequencing system (Applied Biosystems, Inc., l\~ ~ --'19A, ON) by usinga Taq DyeDeoxyTM Tenminator Cycle Sequencing Kit (Applied Biosystems, Inc.).
Template DNA was extracted from ovemight cultures of E. coli DH5~ (pNspX-06) with the WizardsTM minipreps DNA purification system (Promega Corp., Madison, Wl).
Overlapping sequences were generated by primer walking. The DNA sequence data was analyzed using MacDNASlS DNA software (Hitachi Software Engineering Co., Ltd., San Bruno, CA).
DNA structural analysis identified a single 1458 bp open reading frame (ORF), designated xynC, overlapping the Hindlll and Pvull sites of the 3.4 kb EcoRI - Bglll insert and large enough to encode a 51 kDa endo-xylanase (Figure 3). Translation of the ORF would result in the ~ SSiOI1 of a 485 amino acid polypeptide with a predicted molecular weight of 50.4 kDa. Further analyses identified a putative signal sequence (nu~:leotirl~ 301 - 360, Figures 3 and 4) followed by a catalytic domain. The N-tenminal catalytic domain is followed by a putative proline rich, highly reiterated linker region and a region of unknown function.

~ 2~ ~al 94 The extent of the endo-xylanase catalytic domain was determined by deletion analysis. The coding sequence of xynC encoding sequence less the first 41 n~ (nt 301 - 341 Figure 3) was amplified by PCR with oligonucleotide primers Xl(ATCTCTAGAATTCAACTACTCTTGCTCMAG;SEQlDNO.3)andXll(GGG
5 TTG CTC GAG ATT TCT AAT CAA m AT; SEQ ID NO. 4). The oligonucleotides were designed to place an EcoRI site at the 5~ end and a Xhol site at the 3 end of the PCR product. This enabled the xynC PCR product (Figure 4) to be cloned as a I,dnsldliol1al fusion into EcoRI Xhol digested pGEX~T-3 (Phammacia Biotech Inc. Baie D Urie PQ). C;",1,ari,,hi~ colicells lldnb~ulllled with this construct named pGEXxynC, I0 produced endo-xylanase activity. A series of 3 deletions to xynCwas also constructed in pGEX-4T-3 (Figure 5). Fusion proteins (glu ,iu"e S-ll dl ,~r~, dSt,..,~ylanaSe c) were expressed and aflinity purified on glutathione Sepharose 4B according to the GST gene fusion system manual (Phammacia Biotech Inc.). Bound fusion protein was either eluted and used directly in xylanase assays (GST-fusions) or cleaved with thrombin to release 15 only the xyn~encoded peptides. The specific activities of the purified GST-fusions and cleaved xylanase C peptides were d~l~""i"ed. Protein conce~ dliu" was measured with a BioRad (BioRad Ldbul .ies Canada Ltd. 1\~ I~R, ON) protein assay kit.Protein samples were added to a 50 mM potassium phosphate buffer (pH 6.5) containing 1.5 % oat spelt xylan. Samples were incubated at 40~C. Xylanase activity 20 was determined by measuring the amount of reducing sugar released from the substrate (Somogyi 1952). All truncated proteins tested (Figure 5, Table 2) had lower specific acitivities (Table 2) indicating that the full length xylanase C is required for maximal activity. The reduction in activity was particularly pronounced in the case of the Pvu fusion protein. This construct displayed only 1.2% of the activity of the full 25 length GST-XynC fusion protein. By comparison the cleaved Pvu xylanase C protein retained three quarters of the full length protein activity (Table 2).
The specific activity of cleaved affinity purified xylanase C was determined to be 555 units of endo-xylanase activity/mg of protein. One unit of endo-xylanase activity was defined as one umol of reducing sugar equivalents released per minute.

~1 q~l 94 .

Table 2. Relative xylanase activity (~/O) of truncated xylanase C proteins.
Treatment ConstructRelative xylanase activity Cleaved XynC 100.0 l~co47 80.2 Hae 85.3 Pvu 74.8 GST-fusion XynC 100.0 Pvu 1. 2 Example 5. O~,e,~x~,tssiol1 of the Neo,- " "a~li,rpatriciarum endo-xylanase qeneIsolation and chald~ dliol1 of xynC from N. patriciarum 27 enables the large scale production of Xylanase C in any of a number of prokaryotic (e.g., E. co/i and B.
subtilis) or eukayotic (e.g., fungal - Pichia, Saccharomyces, Aspergillus, Trichoderma;
plant - Brassica, Zea, Solanum; or animal - poultry, swine or fish) expression systems using known methods. For example, general teachings for the construction and ~ ssion of xynC in E. coli, P. pastoris, and B. napus are provided below. Similar approaches may be adopted for expression of the N. patriciarum 27 endo-xylanase in other prokaryotic and eukaryotic organisms.
A. Cloning of the Neo " "astix patriciarum xvnC in an Escherichia coli- specific~yl~siol1 construct An expression construct is constructed in which the region encoding the the fulllength xylanase C, less amino acids 1 -14 (Figure 3) is lldns~i,i,uliul1ally fused with the tac promoter (Brosius et al., 1985). The promoter sequences may be replaced by those from other promoters that provide for efficient ~ SSioll in E. coli. The expression construct is introduced into E. colicells by lldl1~r~llllldliull.
I. Construction of the E. co/iexl.l~s~ 1 vector A number of E. coli ~ ssion vectors based on the tac or related promoters are co"""~,~;i..lly available. The constnuct may be prepared with pKK223-3 available from Pharmacia Biotech Inc. The region of xynC encoding the XynC protein (less amino acids 1-14) is amplified with oligonucleotide primers Xlll (SEQ ID NO.5 - GC GM TTC
35 ATG TCA ACT CTT GCT CM AGT TTC) and XIV (SEQ ID NO. 6 - GCC TGC AGT
GAT TTC TM TCA ATT TAT). The oligonuclPoticleS Xlll (SEQ ID NO. 5) and XIV

~ 9~ 9~

(SEQ ID NO. 6) were designed to insert suitable restriction sites at the PCR product's termini to allow direct assembly of the amplified product with pKK223-3. The region of xynCamplified with Xlll (SEQ ID NO.5) and XIV (SEQ ID NO. 6) is digested with EcoRI
and Pstl and ligated into similarly cleaved pKK223-3.
S li. Transformation of E. coliand XylanaseC ~,~,,,tssion The pKK223-3::xynC ligation mix is used to transform competent E coli cells.
Strains suitable for high levels of protein ~c,u~t:SSiO11, such as SG13009, CAG926 or CAG929 (carrying laclon a plasmid such as pREP4), will be employed. T~d~lulll,edcells are spread on LB agar containing ampicillin (100 ,ug/mL) and incubated overnight 10 at 37~C. Ampicillin resistant colonies are screened for the presence of the desired pKK223-3::xynCconstruct by extracting pDNA and subjecting the pDNA to agarose gel electrophoresis and restriction analysis. Positive clones may be further characterized by PCR and nucleotide sequence analysis.
Expression of the N. patriciarum 27 xylanase by 1, dl l~ UU 1 ,ed E. coli cells is tested 15 by growing the cells under vigorous aeration at 37~C in a suitable liquid medium (e.g., LB or 2xYT) containing the d,l)UlU,UI idl~ antibiotic selection until the optical density (600 nm) is between 0.5 and 1Ø The tac promoter is induced by adding isopropyl-~-D-thiogAl~cto~ifle (IPTG) to a final concentration between 0.1 and 2 mM. The cells are cultivated for an additional 2 to 4 h and harvested by centrifugation. Protein ~ sion 20 is monitored by SDS-PAGE, and western bloVimmunodetection techniques.
The expressed XynC may be extracted by breaking (e.g., sonication or mechanical disruption) the E. coR cells. Protein inclusions of XynC may be harvested by centrifugation and solllb" ' with 1 to 2 ~/O SDS. The SDS may be removed by dialysis, electroelution or ulll "" dliOn. The xylanase activity of prepared cell extracts 25 may be assayed by standard methods described in Example 4.
B. Cloning of the Neocallimastix ~atriciarum endo-xylanase in a Pichia pastoris-specific ~,~,u,t,s~ion construct An expression construct is constructed in which the region encoding the the fulllength xylanase C, less amino acids 1 - 14 (Figure 3) is lldnsldliul.al',l fused with the 30 secretion signal sequences found on P. pastoris ex,~ iu" vectors (Pichia Expression Kit Instruction Manual, Invitrogen Corporation, San Diego, CA) in order to express the N. patriciarum xylanase as a secreted product. The promoter and secretion signal 21 90~ 9~

sequences may be replaced by those from other promoters that provide for efficient expression in Pichia. The expression construct is introduced into P. pastoris cells by lld~ UIllldliUII.
i. Construction of the P. pastoris exp~ iu" vector A number of P. pastorls ex~ ,ion vectors based on the aox1 promoters and a-Factor or phol signal sequences are colllll,e,.;i.~'ly available. The construct may be prepared with pPlCaB available from Invitrogen Corporation. The region of xynC
encoding the XynC protein (less amino acids 1-14) is amplified with oligonucleotide primers Xl (SEQ ID NO.3) and Xll (SEQ ID NO. 4). The oligonucleotides Xl (SEQ IDNO. 3) and Xll (SEQ ID NO. 4) were designed to insert suitable restriction sites at the PCR product's termini to allow direct assembly of the amplified product with pPlCaB.
The region of xynC amplified with Xl (SEQ ID NO. 3) and Xll (SEQ ID NO. 4) is digested with EcoRI and Xhol and ligated into similarly cleaved pPlCaB.
ii. Tldll~ull I IdliUn of P. pastoris and XynC ~AUI t:SSiOIl The pPlCaB::xynC ligation mix is used to transform competent E. coli DH5a cells. Tldll~u,,,,edcellsarespreadonLBagarcontainingampicillin(1ûO,ug/mL)and incubated ovemight at 37~C. Ampicillin resistant colonies are screened for the presence of the desired pPlCaB::xynCconstruct by extracting pDNA and subjecting the pDNA to agarose gel el~ulluplloresis and restriction analysis. Positive clones are further ~;l Idl dul~ d by PCR and DNA sequence analysis. Plasmid DNA is preparedfrom a 1 L culture of an E. coliclone carrying the desired pPlCaB::xynC construct. The pDNA is digested with Pmel and analyzed by agarose gel elt:~.l,u~,l,ol~ , to confirm complete digestion of the vector. The digested pDNA is extracted with phenol:chloroform, ethanol plt:~i,uildl~d and resuspended in sterile distilled H20 to a final C(JI~C~31111dliUII of 1 ,ug/,uL. In pl~:lpdldlioll for ildll~ UIllldliUll, P. pastorisGS115 or KM71 cells are grown for 24 h at 30~C in YPD broth. Cells from a 100 ,uL of culture are harvested by centrifugation and resuspended in 100 ,uL of 1, dn~ UI 11 Idliul I buffer (0.1 M
LiCI, 0.1M dillliullllt~ilul, 45~/O polyethylene glycol 4000) containing 10 ,ug salmon spemm DNA and 10 ,ug of linearized pPlCaB::xynC. The mixture is incubated for 1 h at 37~C, spread on YPD agar containing zeocin (1009 ,ug/ml) and incubated for 2 to 5 d at 30~C.
Colonies growing on the selective medium are streaked for purity and analyzed for the presence of the integrated xynC by PCR and Southern blot hybridization.

21 901 q4 Expression of the N. patriciarum 27 xylanase by transfommed P. pastoris cells istested by growing the cells at 30~C and under vigorous aeration in a suitable liquid medium (eg., buffered complex glycerol media such as BMGY) until a culture optical density (600 nm) of 2 to 6 is reached. The cells are harvested and resuspended to an S OD~ of 1.0 in an inducing medium (e.g., buffered complex methanol medium, BMMY) and incubated for a further 3 to 5 days. Cells and cell free culture supennatant are collected and protein ~ ssi.,n is monitored by enzyme assay, SDS-PAGE, and westem blot/immullod~ ,liol1 techniques.
C. Clonina of the Neocallimastix patriciarum endo-xylanase in a Brassica rlaPus seed 10 - sPecific t"~pl~5iOI1 construct Transfommation and gene ~ plt~ iOIl methods have been developed for a wide variety of monocotyledonous and dicotyledonous crop species. In this example, a N.
patriciarum 27 xylanase ~ ssi~n constnuct was constructed in which the region encoding the full length xylanase C, less amino acids 1 - 14 (Figure 3) is lld~lsldli~Jnally~5 fused with an oleosin coding sequence in order to target seed oil body specific ssion of the N. patriciarum xylanase. The promoter and/or secretion signal sequences may be replaced by those from other promoters that provide for efficient ~xp~s~io" in B napusoranyotherl~dl~ ""ableplantspecies in ordertoachievethe same goal as is the objective of this invention. The ~ 5:,;0n constnuct is introduced 20 into B. napus cells by Agrobacterium mediated transfommation.
L constructiQn of the B, naDus ~ s~ion vector A number of ~X~J,t,ssiol1 vectors functional in B. napus are described in the literature (Gelvin et al., 1993). To constnuct a oleosin-xylanase gene fusion, the oleosin and l~colllbil1dll1 xynCgenes were first cloned into pBluescriptllKS+ (pBS) to creat an 25 illL~Illledidl~ plasmid. The construct pCGYOBPGUSA (van Rooijen and Moloney, 1995b) was digested with Pstl and BamHI to isolate the 1608-bp fragment containing the oleosin promoter and oleosin coding region. The xynC coding region was obtained by the digestion of pGEXxynC with BamHI and Xhol. These two fragments were cloned into pBS previously digested with Pstl and Xhol. The resulting plasmid was de:,iy, Idl~d 30 as pBSOleXyn. To obtain a nopaline synthetase (NOS) temminator sequence flanked by Xbal and Xhol restriction sites, a BamHI and Hinalll fragment from pCGYOBPGUSA
was subcloned into pBS to make an i"l~l"~edidl~ plasmid pBSNos. Digestion of this ~1 9~1 9~
constnuct with Xba I and Xhol liberated a Xbal Xhol flanked fragment containing the NOS terminator. The oleosin-xylanase C t~,ult:55iOIl construct was assembled in pCGN1559 (McBride and Summerfelt 1990). The oleosin-xynCgene fusion was cut out of pBSOleXyn with Pstl and Xhol. This fragment was ligated with the Xbal Xhol 5 flanked NOS fragment from pBSNos and Pstl Xhol digested pCGN1559. This plasmid was named pCGOleXyn.
ii. Trdl i~ionl ,aliu" of B. napus and stablç xylanase Ç ~Xu~55iOIl Transgenic B. napus were prepared as described by van Rooijen and Moloney (1995a; 1995b). Agrobacterium tumefaciens strain EHA101 was transfommed by 10 ~ Iu,uoldtiùll with pCGOleXyn. Cotyledonary petioles of B. napuswere tldll~iul",ed with A. t~",~r~ "s EHA101 (pCGOleXyn). Transgenic plants were regenerated from explants that rooted on hormone-free MS medium containing 20 ug/mL kanamycin.
Young plants were assayed for NPTII activity grown to maturity and allowed to self pollenate and set seed. Seeds from individual lld":,~unllalll~ were pooled and the l5 presence of ~¢ynC was confirmed by PCR and Southem blot hyl ri- i~dlion. XynCproduction was confimmed by westem blot immu, lodt~l~ulioll with polyclonal antibodies specific for this protein. Part of the seed sample was assayed for xylanase activity, and compared to seeds from u"l, dl ,:,~u, " ,ed plants (Table 3). Oil bodies were isolated from mature dry seeds by the method described in van Rooijen and Moloney (1 995a). Oil 20 bodies were suspended in 50 mM potassium pho:,uhdl~ buffer (pH 4.5). Xylanaseassays were perfommed as described in Example 4. Transgenic plants carrying the oleosin-xylanase C constnuct produced 10 to 50 times higher levels of xylanase activity than the wild type control plants.
The -rr~ ~- 1 of l,dnsg~,,icoil-bodiesasani""": dmatrixwastestedas 25 described in van Rooijen and Moloney (l 995a). Oil-bodies carrying oleosin-xylanase C fusion protein (0.1 mL) were mixed with 0.2 mL of substrate mix containing 0.5~/O
RBB-xylan in a 50 mM potassium-phosphate buffer (pH 6.5). The reaction mixtures were incubated for 60 min at 40~C. After each incubation, the reaction mix was centrifuged to separate oil-bodies and substrate. The ~u~ mdldl ,I was then removed 30 and the oil pad was recycled in a new reaction through the addition of fresh substrate.
The ulllt:llldldlll was assayed for RBB-xylan digestion by the method of Biely et al.
(1988). Absolute ethanol (0.8 mL) was added to the u"l~i, laldn t samples to stop the 21 9~ ~4 xylanase reactions. The samples were allowed to stand at room temperature for 30 min and centrifuged for 5 min in a microfuge to remove the precipitated substrate. The absoi bal1ces of the resulting suu~" Idld~ were measured at 595 nm. The transgenic oil-bodies retained their endo-xlanase activity through four rounds of recycling (Table 5 4). These results clearly cl~lllon~LIdl~d the stability and potential of lldl15yt"~ic oil-bodies as an i""" ' ~ enzyme matrix.

Table 3. Xylanase activity of ~, . ., "1,; "~ oil-bodies extracted from transgenic canola carrying the oleosin-xylanase C expression construct.
Transgenic line Xylanase activity nmol/min/mg (standard error) Wild type 0.413 (0.31) Tl 18.78 (2.63) T4 29.19 (8.39) T7 2Z.85 (0.42) T13 6.68 (0.76) T18 12.23 ( I .74) T23 5.86 (0.84) Table 4. Xylanase activity of recycled oil-bodies Number of cycles Relative xylanase activity 100.0 2 152.5 4 148.5 Example 6. Ide, I~; icdliùl I of Related Xylanase Genes in Other Microorganisms To identify a xylanase gene related to xynC, hylJIidi~dliùll analysis can be used 35 to screen nucleic acids from other organisms of interest using xynC (SEQ ID NO. 1 ) or portions thereof as probes by known techniques (Ausubel et al., 1990, Sambrook et al., 1989). Related nucleic acids may be cloned by employing techniques known to those skilled in the art. R~l;uisu~ .es (i.e., 32p) may be required when screening olyani~",s with complex genomes in order to increase the sensitivity of the analysis. Polymerase 40 Chain Reaction (PCR) a" ,, ' " ~ .1 may also be used to identify genes related to xynC.

Related sequences found in pure or mixed cultures are p~ tn it;all~l amplified by PCR
(and variations of such as Reverse Transcription - PCR) with oligonucleotides primers designed using SEQ ID NO. 1. Amplified products may be visualized by agarose gelelectrophoresis and cloned using techniques know to those skilled in the art.
A variety of materials, including cells, colonies, plaques, and extracted nucleic acids (e.g., DNA, RNA), may be examined by these techniques for the presence of related sequences.
All pll' I ,~ ",el,lioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All p~' I " ,s are herein illcollJoldl~d by reference to the same extent as if each individual publication was :".e.;i~ica'ly and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and examples for purposes of clarity and of understanding, it will be obvious that certain changes and mo~ ns may be practised within the scope of the appended claims.

REFCRCrlCES

Ausubel, F.A., R. Brent, R.E. Kingston, D.D. Moore, J.G. Sneidman, J.A. Smith, and K.
Struhl. (eds.) 1990. Current protocols in molecular biology. Green Publishing and Wiley-l"l~,~ciel1ce, New York.
Biely, P., D. Mislovicova and R. Toman. 1988. Remazol brilliant blue-xylan: a soluble cl ll umoyt:l lic substrate for xylanases. Methods Enzymol. 160:536-542.
Brosius, J., M. Erfl and J. Storella. 1985. Spacing of the -10 and -35 regions in the tac promoter. J. Biol. Chem. 260:3539-3541.
I0 Chesson, A., C.W. Forsberg, and E. Grenet. 1995. Improving the digestion of plant cell walls and fibrous feeds. In: M. Joumet, E. Grenet, M-H. Farce, M. Theriez, C.
Demarquilly (eds) Recent develo,ul "enl~ in the nutrition of herbivores. P, ucde.li, ,y~
of the IVth l"l~" IdliUI Idl Symposium on the Nutrition of Herbivores. INRA Editions, Paris. pp249-277.
Ellis, S.B., P.F. Brust, P.J. Koutz, A.F. Waters, M.M. Harpold, and R.R. Gingeras.1985.
Isolation of Alcohol oxidase and two other methanol regulated genes from the yeast, Pichia pastoris. Mol. Cell. Biol. 5:1111-1121.
Gelvin, S.B., R.A. Schilperoort, and D.P.S. Verma. (eds.).1993. Plant Molecular Biology Manual. Kluwer Academic Publishers, Boston, MA.
Hodgson J. (1994) The changing bulk biocatalyst market. Bio/Technology 12: 789-790 Lowe, S.E., M.K. Theodorou, A.P. Trinci, and R.B. Hespell. (1985) Growth of anaerobic fungi on defined and semi-defined media lacking rumen fluid. J. Gen. Microbiol.
131 :2225-2229.
McBride, K.E. and K.R. Summerfelt.1990. Improved binary vectors for Aylvba~luli~lm mediated plant llal1~ulllldlium Plant Mol. Biol. 15:269-276.
McNeil M., A.G. Darvill, S.C. Fry and P. Albersheim. (1984) Structure and function of the primary cell wall of plants. Ann Rev Biochem 53:625-663 Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular clonin~. A laboratory manual. 2nd. edn. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY.
Somogyi, M.J. 1952. Notes on sugar determination. J. Biol. Chen. 195:19-23.

Tamblyn Lee, J.M., Y. Hu, H. Zhu, K.-J. Cheng, P.J. Krell and C.W. Forsberg. 1993.
Cloning of a xylanase gene from the ruminal fungus NeQ. - " "a:,l;X patriciarum 27 and its ex~ ," in Es~,l,oli,,l)id coli. Can J. Microbiol. 39:134-139.
Teather, R.M. and P.J. Wood.1982. Use of Congo red -poly~ac.,l,a~ ld~;liolls in S enumeration and chald~ dlion of cellulolytic bacteria from the bovine rumen.
Appl. Environ. Microbiol. 43:777-780.
van Rooijen, G.J.H. and M. M. Moloney. 1995a. Plant seed oil-bodies as carriers for foreign proteins. Bio/Technology 13:72-77.
van Rooijen G.J.H. and M.M. Moloney. 1995b. Structural requirements of oleosin domains for sl Ihcelll llAr targeting to the oil body. Plant Physiol 109:1353-1361 Wong, S.-L. 1989. Development of an inducible and enhancible expression and secretion system in Bacillus subtilis. Gene 83:215-223.

2 ~ 9 4 .

SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: Cheng, Kuo-Joan Selinger, Leonard B.
Liu, Jin-Hao Hu, Youji Forsberg, Cecil W.
Moloney, Maurice M.
(ii) TITLE OF INVENTION: A xylanase obtained irom an anaerobic ~ungus (iii) NUMBER OF SEQUENCES: 6 (iV) ~KK~UN~N~ ADDRESS:
(A) AnnR~ T~T~: McKay-Carey & Company (B) STREET: 2125 Commerce Place, 10155 - 102nd Street (C) CITY: Edmonton (D) STATE: Alberta (E) COUNTRY: Canada (F) ZIP: TSJ 4G8 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: McKay-Carey ~ Company, (ix) TRT~R~nMMTTT\TTcATIoN INFORMATION:
(A) TELEPHONE: (403) 424-0222 (B) TELEFAX: (403) 421-0834 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2058 base pairs (B) TYPE: nucleic acid (C) STRAT~TnRnN~ double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~ lCAL: NO

(iv) ANTI-SENSE: NO

30~

21 9ûl 94 .

(vi) ORIGINAL SOURCE:
(A) ORGANISM: Neocallimastix patriciarum (B) STRAIN: 27 (vii) IMMEDIATE SOURCE:
(A) LIBRARY: genomic DNA library (B) CLONE pNspX-06 (ix) PEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 301 1755 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
ATATTATAAT AATTGTTCAA AAAAAGTAAT AA~AAAAAAA AAATTTTTTT T1L111L111 60 GGGAAAATTG AGTATAaATA ~lll~ r TACCTTTTTT ~lllll~l TTATTCTTTA 120 TATTTATTGG AATTATTTAC TTTCACTGGT ~.~.AAA~AAA~ ATTAATAGTG ~A~AA~A~AT 240 TATTAGAAAA A~.AAAAAAAA AAATTATTAC AATTAATTAC ~A~AAA~AAA ATAGTTAAaA 300 ATG AaA TTT TTA CAA ATT ATT CCT GTA TTA TTA TCT TTA ACT TCA ACT 348 Met Lys Phe Leu Gln Ile Ile Pro Val Leu Leu Ser Leu Thr Ser Thr =~

Thr Leu Ala Gln Ser Phe Cys Ser Ser Ala Ser His Ser Gly Gln Ser Val Lys Glu Thr Gly Asn Lys Val Gly Thr Ile Gly Gly Val Gly Tyr Glu Leu Trp Ala Asp Ser Gly Asn Asn Ser Ala Thr Phe Tyr Ser Asp 50 55 60 '-Gly Ser Phe Ser Cys Thr Phe Gln Asn Ala Gly Asp Tyr Leu Cys Arg Ser Gly Leu Ser Phe Asp Ser Thr Lys Thr Pro Ser Gln Ile Gly Arg ATG AAG GCT GAT TTC AaA CTT GTC AaA ACA AAA TAT TTC CAA TGT TGG 636 Met Lys Ala Asp Phe Lys Leu Val Lys Thr Lys Tyr Phe Gln Cys Trp 100 105 110 =.

Leu Phe Leu Cys Trp Cys Leu Arg Trp Thr Arg Ser Pro Leu Val Gly 3o~

2~ 90~ 94 .

Ile Leu His Val Asp Asn Trp Leu Ser Pro Ser Pro Pro Gly Asp Trp Val Gly Asn Lys Lys His Gly Ser Phe Thr Ile Asp Gly Ala Gln Tyr Thr Val Tyr Glu Asn Thr Arg Thr Gly Pro Ser Ile Asp Gly Asn Thr ACC TTC AAA CAA TAC TTT AGT ATT CZT CAA CA~ GCT CGT GAT TGT GGT 876 Thr Phe Lys Gln Tyr Phe Ser Ile Arg Gln Gln Ala Arg Asp Cys Gly Thr Ile Asp Ile Ser Ala His Phe Asp Gln Trp Glu Lys Leu GIy Met Thr Met Gly Lys Leu His Glu Ala Lys Val Leu Gly Glu Ala Gly Asn 210 2~5 220 Gly Asn Gly Gly Val Ser Gly Thr Ala Asp Phe Pro Tyr Ala Lys Val Tyr Ile Gly Asp Gly Asn Gly Gly Gly Ala Ser Pro Ala Pro Ala Gly ::

Gly Ala Pro Ala Gly GIy Ala Pro AIa Gly Asn Asp Gln Pro Gln Gly ~ ~~ 260 265 270 Pro Gln Gly GIn Gln Pro Pro Gln Gly Gln Gln Pro Pro Gln Gly Gln 275 280 285 ~:

Gln Pro Pro Gln Gly Gln Gln Pro Pro Gln Gly Gln Gln Pro Pro Gln 290 2g5 300 Gly Asn Asp Gln Gln Gly Gln Gln Pro Pro Gln Gly GIn GIn Pro Pro ::
305 310 = 315 320 CAA GGT AAC GAT CAA CAA CAA GGA CAA CAA CCA CCA CAA CCA C~A GGA 1308 Gln Gly Asn Asp Gln Gln Gln Gly Gln Gln Pro Pro Gln Pro Gln Gly Pro Gln Gly Gly Asn Pro Gly Gly Ser Asp Phe Asn Asn Trp Asn Gln '~

3c c .

Gly Gly Ser Pro. Trp Gly Gly Asn Gln GIy Gly Ser Pro Trp Gly Gly Asn Gln Gly Gly Asn Pro Trp Gly Gly Asn GIn Gly Gly Ser Pro Trp 370 375 380 :

Gly Gly Asn Gln Gly Gly Ser Pro Trp Gly Gln Gly Asn Gln Gly Gly 385 390 395 . 400 Asn Pro Trp Gly Gly Asn Gln Gly Gly Ser Pro Trp Gly Gly Asn Gln GGT GGT AAT CCA TGG GGT GGT AAT CAA TGG GGT GCT CCA CA~ AAT GCT 1596 Gly Gly Asn Pro Trp Gly Gly Asn Gln Trp Gly Ala Pro Gln Asn Ala Ala Ala Pro Gln Ser Ala Ala Ala Pro Gln Asn Ala Ser Asp Gly Gly Asn Cys Ala Ser Leu Trp Gly Gln Cys Gly Gly Gln Gly Tyr Asn Gly Pro Ser Cys Cys Ser Glu Gly Ser Cys Lys=Pro Ile Asr Glu Tyr Phe His Gln Cys Gln Lys TAGATTAAAA TAATAAA~.AA AAAAAAAAAA AL1LL1L1L~ lLl~LLLlLL LLLLl~ 1855 CAATTAATAA ATCATTAAAA TAGATCATTA ATATAATTAT TTATTTTCAT ;L L 1 L LLLll 1915 ATAAAA~.ATA CTATTTTAAT AAAATTATAA AAAAAAAATA ~ATAAAAAAA AAATATAAAA 2035 (2) INFORNATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS: =
(A) LENGTH: 485 amino acids (B) TYPE: aAlino acid (D) TOPOLOGY: linear (ii) NOLECULE TYPE: protein 3o ~

21 901 't~

(xi) SEQ~7ENCE DESCRIPTION: SEQ ID No:2:
~et Lys Phe Leu Gln Ile Ile Pro Val Leu Leu Ser Leu Thr Ser Thr ~hr Leu Ala Gln Ser Phe Cy6 Ser Ser Ala Ser His Ser Gly Gln Ser Val Lys Gru Thr Gly Asn Lys Val Gly Thr Ile Gly Gly Val Gly Tyr Glu Leu Trp Ala Asp Ser Gly Asn Asn Ser Ala Thr Phe Tyr Ser Asp Gly Ser Phe Ser Cys Thr Phe Gln Asn Ala Gly Asp Tyr Leu Cys Arg ~er Gly Leu Ser Phe Asp Ser Thr Lys Thr Pro Ser Gln Ile Gly Arg ~et Lys Ala Asp Phe Lys Leu Val Lys Thr Lys Tyr Phe Gln Cys Trp 100 105 ~ 110 Leu Phe Leu Cys Trp Cys Leu Arg Trp Thr Arg Ser Pro Leu Val Gly Ile Leu His Val Asp Asn Trp Leu Ser Pro Ser Pro Pro Gly Asp Trp Val Gly Asn Lys Lys His Gly Ser Phe Thr Ile Asp Gly Ala Gln Tyr ~hr Val Tyr Glu Asn Thr Arg Thr Gly Pro Ser Ile Asp Gly Asn Thr ~hr Phe Lys Gln Tyr Phe Ser Ile Arg Gln Gln Ala Arg Asp Cys Gly Thr Ile Asp Ile Ser Ala His Phe Asp Gln Trp Glu Lys Leu Gly Met Thr Met Gly Lys Leu His Glu Ala Lys Val Leu Gly Glu Ala Gly Asn Gly Asn Gly Gly Val Ser Gly Thr Ala Asp Phe Pro Tyr Ala Lys Val 225 230 235 2g0 ~yr Ile Gly Asp Gly Asn Gly GIy Gly Ala Ser Pro Ala Pro Ala Gly ~ly Ala Pro Ala Gly Gly Ala Pro Ala Gly Asn Asp Gln Pro Gln Gly 260 265 ~ 270 Pro Gln Gly Gln Gln Pro Pro Gln Gly Gln Gln Pro Pro Gln Gly Gln 3~ F

21 9~1 9~

Gln Pro Pro Gln Gly Gln Gln Pro Pro Gln Gly Gln Gln Pro Pro Gln Gly Asn Asp Gln Gln Gly Gln Gln Pro Pro Gln Gly Gln Gln Pro Pro r ~ln Gly Asn Asp Gln Gln Gln Gly Gln Gln Pro Pro Gln Pro GIn Gly ~ro Gln Gly Gly Asn Pro Gly Gly Ser Asp Phe Asn Asn Trp Asn Gln Gly Gly Ser Pro Trp Gly Gly Asn Gln Gly Gly Ser Pro Trp Gly Gly Asn Gln Gly Gly Asn Pro Trp Gly Gly Asn GIn Gly Gly Ser Pro Trp Gly Gly Asn Gln Gly Gly Ser Pro Trp Gly Gln Gly Asn Gln Gly Gly ~sn Pro Trp Gly Gly Asn Gln Gly Gly Ser Pro Trp Gly Gly Asn Gln ~ly Gly Asn Pro Trp Gly Gly Asn Gln Trp Gly Ala Pro Gln Asn Ala Ala Ala Pro Gln Ser Ala Ala Ala Pro Gln Asn Ala Ser Asp GIy Gly Asn Cys Ala Ser Leu Trp Gly Gln Cys Gly Gly Gln Gly Tyr Asn Gly Pro Ser Cys Cys Ser Glu Gly Ser Cys Lys Pro rle Asn GIu Tyr Phe ~is Gln Cys Gln Lys ~2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) sTR~mFn~Fc~: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oligonucleotide XI~' (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

21 901 ~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE ~TTARA~TERT.CTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) s~RANnEnNT~sc: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "oliyonucleotide XII"
(iii) ~Y~u~ CAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
;LL\~ C~; AGATTTCTAA TC~TTTAT 293 : :
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) s~RAT\TnEnTTEcs single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = ~oligonucleotide XIII" :~
(iii) ~Y~ CAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GCGAATTCAT GTCAACTCTT GCTCAAAGTT TC .. . 32 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE ~TTARA~TERTCTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) sTRAT\TnEnT\T~cc: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid 30 Gi 2~ 90~ q4 (A) DESCRIPTION: /desc = "oligonucleotide ~rv"
(iii) ~YS~~ CAL: NO
(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

~0 ~

Claims (21)

1. A purified and isolated DNA comprising a xylanase coding region that encodes a xylanase obtained from a strain of the species Neocallimastix patriciarum.

2. A purified and isolated DNA according to claim 1 wherein the xylanase coding region is capable of hybridizing under conditions of medium stringency with a probe comprising at least 25 continuous nucleotides of nucleotide sequence SEQ
ID NO. 1.

3. A purified and isolated DNA according to claim 2, wherein the encoded xylanase comprises amino acid sequence SEQ ID NO. 2.

4. A purified and isolated DNA according to claim 3, wherein the xylanase codingregion comprises nucleotides 301-1755 of nucleotide sequence SEQ ID NO. 1.

5. A purified and isolated DNA according to claim 2, wherein the xylanase codingregion is operably linked to control sequences capable of effecting expression of the coding region in a suitable expression host.

6. A purified and isolated DNA according to claim 5, wherein at least one of the control sequences is heterologous to the xylanase coding region.

7. A purified and isolated DNA according to claim 2 further comprising an oleosin coding region that encodes an oleosin protein, said oleosin coding region operably linked to the xylanase coding region.

8. A purified and isolated xylanase obtained from a strain of the species Neocallimastix patriciarum.

9. A purified and isolated xylanase according to claim 8 wherein the xylanase isencoded by a DNA capable of hybridizing under conditions of medium stringency with a probe comprising at least 25 continuous nucleotides of nucleotide sequence SEQ ID NO. 1.

10. A purified and isolated xylanase according to claim 9 wherein the xylanase comprises amino acid sequence SEQ ID NO. 2.

11. A method for producing a xylanase, comprising the steps of:
(a) transforming at least one host cell with a DNA according to claim 2 so that the host cell can express a xylanase encoded by the DNA; and (b) growing a culture of the host cells under conditions conducive to the expression of the encoded xylanase by the host cells.

12. A host cell transformed with a DNA according to claim 2 so that the host cell can express a xylanase encoded by said DNA.

13. A transgenic plant transformed with a DNA according to claim 2 so that a xylanase encoded by the DNA can be expressed by the plant.

14. A transgenic plant according to claim 13 wherein the plant is of the species Brassica napus.

15. A transgenic plant according to claim 14 wherein the plant is transformed with a DNA encoding a oleosin-xylanase fusion protein so that the oleosin-xylanase fusion protein encoded by the DNA can be expressed by the plant, said DNA
comprising a xylanase coding region capable of hybridizing under conditions of medium stringency with a probe comprising at least 25 continuous nucleotides of nucleotide sequence SEQ ID NO. 1, and an oleosin coding region that encodes an oleosin protein, said oleosin coding region operably linked to the xylanase coding region.

16. A method for producing a transgenic plant capable of expressing a xylanase, comprising the steps of:
(a) transforming a plant with a DNA according to claim 2 so that the plant can express a xylanase encoded by the DNA; and (b) growing the plant under conditions conducive to the expression of the encoded xylanase by the plant.

17. A method for producing a transgenic plant according to claim 16, comprising the steps of:
(a) transforming a plant with a DNA encoding an oleosin-xylanase fusion protein so that the oleosin-xylanase fusion protein encoded by the DNA can be expressed by the plant, said DNA comprising a xylanase coding region capable of hybridizing under conditions of medium stringency with a probe comprising at least 25 continuous nucleotides of nucleotide sequence SEQ ID NO. 1 and an oleosin coding region that encodes an oleosin protein, said oleosin coding region operably linked to the xylanase coding region; and (b) growing the plant under conditions conducive to the expression of the encoded oleosin-xylanase fusion protein by the plant.

18. A feed supplement comprising a xylanase according to claim 9.

19. A method for identifying a nucleic acid molecule from an organism, said nucleic acid molecule encoding a xylanase, said method comprising the steps of:
(a) isolating nucleic acid molecules from the organism; and (b) performing nucleic acid hybridization under conditions of medium to high stringency with nucleic acid molecules and a labelled hybridization probe havinga nucleotide sequence comprising at least 25 continuous nucleotides of SEQ ID
NO. 1.

20. A method according to claim 19 wherein the hybridization conditions are of medium stringency.

21. A method according to claim 20 wherein the hybridization conditions are of high stringency.