NZ252937A

NZ252937A - Cloning recombinant xylanase from anaerobic rumen fungus

Info

Publication number: NZ252937A
Application number: NZ25293793A
Authority: NZ
Inventors: Gang Ping Xue
Original assignee: Commw Scient Ind Res Org
Priority date: 1992-06-17
Filing date: 1993-06-17
Publication date: 1996-10-28
Also published as: EP0746607A1; CA2138399A1; WO1993025671A1; EP0746607A4; FI945889A0; FI945889A; BR9306576A; JPH07508647A

Description

<div class="application article clearfix" id="description"> New Zealand No. 252937 International No. PCT/AU93/00294 j r io<;ty Dats(s): .3S.).!r|.S.3.. | C-smptote SpociftCatten Filed: ...Q|.!?.i.O. | Ok*: (QtorAv3|^fe3;.Cri9f.?J..|oo,l 9ob^-sasSon Dfeta: .2..U...0.CT...1996. 3.0. Jour-*! No: NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION m Title of Invention: Recombinant xylanase t Name, address and nationality of applicant(s) as in international application form: a* A*jV<*ii COMMONWEALTH SCIENTIFIC & INDUSTRIAL RESEARCH ORGANISATION of Limootono Avonuo, Campboll; Canborro 2601, Auotrolion Capitol Torritory, Australia 4oT flb»«rk.vt||< ( V!cf-or?a JoSt, 93/25671 PCT/AU93/00294 25293 1 TITLE "RECOMBINANT XYLANASE" FIELD OF INVENTION This invention relates to a recombinant xyianase derived from an 5 anaerobic fungus and a method of production of the recombinant xyianase and clones utilised in the method. BACKGROUND ART Xylan is a major component of hemicellulose and the second major component of plant fibre. Xylan consists of a backbone of /?-1,4-linked 10 xylose units. The enzymic cleavage of 12.-1,4-xylosidic linkages is performed by endo-B-1,4-xylanases (xylanases). Many microorganisms produce extracellular xylanases. In the past decade, many xyianase genes were isolated from lignoceliulolytic bacteria, but isolation of xyianase genes from fungi with functional expression in E. coli has not 15 been documented prior to this invention. Lignoceliulolytic fungi usually produce more active xyianase than bacteria, in particular, the anaerobic fungus NeocalJimastix patriciarum, isolated from the sheep rumen, has a high capacity for xylan degradation. 20 Reference may also be made to other prior art which serves as background prior art prior to the advent of the present invention. Such prior art includes: (i) Reymond et. al. Gene 110 (1992) 57-63; (ii) Wong et. al. Clin. Reviews in Biotechnology 12 413-435 25 (1992); (iii) Orpin et. al. Current Microbiology Vol 3 (1979) pp 121-124; (iv) Mountfort and Asher in "The Roles of Protozoa and Fungi in Ruminant Digestion" (1989) Pernambul Books (Australia); 30 (v) Joblin et. al. FEMS Microbiology Letters 65 (1989) 119- 122; WO 93/25671 PCT/AU93/00294 2 (vi) Lowe et. al. Applied and Environmental Microbiology June 1987 pp 1210-1215; and (vii) Lowe et. al. Applied and Environmental Microbiology June 1987 pp 1216-1223. 5 Cloning of xyianase genes from bacteria can be achieved by isolation of enzymatically active clones from genomic libraries established in E. coli. However this .- pproach for isolation of xyianase genes from fungal genomic libraries with functional expression of xyianase is not possible. This is because fungi are eucaryotic 10 microorganisms. Most eucaryotic genes contain introns and E. coli is unable to perform post-transcriptional modification of mRNAs in order to splice out introns. Therefore, enzymatically functional protein cannot normally be synthesised in clones obtained from a fungal genomic library. 15 The cDNA cloning approach can be used to overcome the post- transcriptional modification problem in E. coli. However, xylanases in fungi are usually glycosylated and glycosylation is often required for biological activity of many glycosylated enzymes. E. coli lacks a glycosylation mechanism. This problem can be solved if the cloned gene 20 is transferred to an eucaryotic organism, such as yeast. Other problems which are often encountered in obtaining a biologically functional protein from a cDNA clone in E. coii are (i) that many eucaryotic mRNAs contain translational stop codons upstream of the translational start codon of a gene which prevents the synthesis of the cloned protein from the 25 translational start provided in the vector, and (ii) that synthesis of the cloned protein is based on fusion proteins and the biological function of the cloned protein is often adversely affected by the fused peptide derived from the cloning vector. Therefore, in the past, researchers in this field employed 30 differential or cross hybridisation, antibody probes or oligonucleotide probes for the isolation of fungal polysaccharide hydrolase cDNA or genomic DNA clones. Relevant publications in this regard include WO 93/25671 PCT/AU93/00294 3 Reymond et. al. FEMS Microbiology letters 77 (1991) 107-112; Teeri et. al., Biotechnology 1 696-699 (1983); Shoemaker et. al., Biotechnology 1 691-696 (1983); Sims et. al. Gene 74 411-422 (1988); Morosoli and Durand FEMS Microbiology Letters 5J_ 217-224 5 (1988); and Azevedo et. al. in J. Gen. Microbiol. 136 2569-2576 (1990). However, these methods are very time-consuming, and quite often two stages of intensive cloning work are required for isolation of an enzymatically functional clone. For antibody or oligonucleotide probes, purification of the fungal xyianase is also required. It usually 10 takes more than one year to obtain a functional enzyme clone using the above approaches. Isolation of fungal xyianase cDNAs by utilising an expression system in E. coli has not been reported prior to the advent of this invention probably at least partially due to failure in obtaining 15 enzymatically functional xyianase clones by using improper expression vectors. Selection of expression vector systems is important. If plasmid expression vectors such as pUC vectors are used, and the cloned enzyme is trapped inside the cell, therefore screening for xyianase clones by the convenient xylan-agar plate technique is difficult. Bacteriophage 20 vectors have an advantage in respect to the release of the cloned enzyme into xylan-agar medium due to cell lysis. However, commonly used bacteriophage expression vectors, /Igtl 1 and its derivatives, have polyclonal sites at the C-terminus of the LacZ peptide. The large part of LacZ peptide fused to the cloned enzyme often adversely affects the 25 cloned enzyme activity. In specific regard to the abovementioned Reymond et. al. (1991) reference there is described an attempt of molecular cloning of polysaccharide hydrolase (ie. cellulase) genes from an anaerobic fungus which is N. frontalis. In this reference a clone from a cDNA library 30 derived from N. frontalis hybridized to a DNA probe encoding part of the exo-cellobiohydrolase (CBH 1) gene of Trichoderma reesei. However it was subsequently revealed by Reymond et. al. in a personal WO 93/25671 PCT/AU93/00294 4 communication that the particular cDNA clone obtained from N. frontalis does not encode any polysaccharide hydrolase. Moreover the Reymond et. al. reference did not describe the production of biologically functional enzymes from these clones. 5 In relation to isolation of a fungal xyianase gene, the only report that exists so far prior to this invention is the abovementioned Morosoli and Durand reference which describes isolation of a xyianase gene from yeast Crvptococcus albidus using differential hybridization techniques. However, this reference does not describe the production of biologically 10 functional enzymes from this xyianase gene. BROAD STATEMENT OF INVENTION It is an object of the invention to provide a recombinant xyianase from an anaerobic rumen fungus which may be of use commercially in relation to hydrolysis of xylan. 15 A further object of the invention is to provide a method of cloning of xyianase cDNAs from an anaerobic rumen fungus which may encode the recombinant xyianase of the invention. A further object of the invention is to provide xyianase clones which may be produced in the abovementioned method. 20 The method of cloning of the invention includes the following steps: (i) cultivation of an anaerobic rumen fungus; (ii) isolating total RNA from the culture in step (i); (iii) isolating poly A+ mRNA from the total RNA referred to in 25 step (ii); (iv) constructing a cDNA expression library; (v) ligating cDNAs to a bacteriophage expression vector selected from AZ&P, AZAP II or vectors of similar properties; 30 (vi) screening of xyianase positive recombinant clones in a culture medium incorporating xylan by detection of xylan hydrolysis; and WO 93/25671 PCT/AU93/00294 5 (vii) purifying xyianase positive recombinant clones. In step (i) above in relation to preparation of the recombinant xyianase, from anaerobic fungi, particularly alimentary tract fungi, may be cultivated as described hereinbelow. These fungi are strict anaerobes 5 and may be exemplified by Neoca/limastix patriciarum, Neocaf/imastix frontalis, Neoca/limastix hurleyensis, Neocallimastix stanthorpensis, Sphaeromonas communis, Caecomyces equi, Piromyces communis, Piromyces equi, Piromyces dumbonica, Piromyces lethargicus, Piromyces mai, Ruminomyces elegans, Anaeromyces mucronatus, Orpinomyces 10 bovis and Orpinomyces joyonii. In regard to the above mentioned anaerobic alimentary tract fungi, Caecomyces equi, Piromyces equi, Piromyces dumbonica and Piromyces mai are found in horses and thus are not located in the rumen of cattle like the other fungi described above. 15 The cultivation may proceed in appropriate culture media containing rumen fluid and also may contain cellulose such as Avicel (ie. a form of microcrystalline cellulose) as a carbon source under anaerobic conditions. After cultivation of the fungi total RNA may be obtained in any suitable manner. Thus initially the fungal cells may be harvested by 20 filtration and subsequently lysed in appropriate cell lysis buffer by mechanical disruption. A suitable RNA preserving compound may also be added to the fungal cells to maintain the RNA intact by denaturing RNAses which would otherwise attack the fungal RNA. The total RNA may subsequently be isolated from the homogenate by any suitable 25 technique such as by ultracentrifugation through a CsCI2 cushion or alternative technique as described by Sambrook et. al. in Molecular Cloning; A Laboratory Manual 2nd Edition Cold Spring Harbor Laboratory Press in 1989. An alternative method for preparation of total fungal RNA to that described above may be based on or adapted from 30 the procedure described in Puissant and Houdebine in Bio-Techniques 148-149 in 1990. Total fungal RNA in this alternative technique may also be isolated from the above homogenate by extraction with phenol WO 93/25671 PCT/AU93/00294 6 chloroform at pH4 to remove DNA and associated protein. Total RNA obtained was further purified by washing with lithium chloride-urea solution. Poly (A) + mRNA may then be isolated from the total RNA by 5 affinity chromatography on a compound containing multiple thymine residues such as oligo (dT) cellulose. Alternatively a compound containing multiple uracil residues may be used such as poly (U)-Sephadex. The poly (A)+ mRNA may then be eluted from the affinity column by a suitable buffer. 10 A cDNA expression library may then be constructed using a standard technique based on conversion of the poly (A)+ mRNA to cDNA by the enzyme reverse transcriptase. The first strand of cDNA may be synthesised using reverse transcriptase and the second strand of the cDNA may be synthesised using E. coli DNA polymerase I. The cDNA 15 may subsequently be fractionated to a suitable size and may be ligated to the bacteriophage expression vector, preferably /IZAP or AZAPII. The cDNA library may then be amplified after packaging in vitro, using any suitable host bacterial cell such as a suitable strain of E. coli. The choice of the bacteriophage expression vector in step (v) is 20 important in that such expression vector should include the following features: (i) having an E. coli promoter; (ii) having a translation start codon; (iii) having a ribosomal binding site; 25 (iv) the fusion peptide derived from the vector should be as small as possible as the biological function of the cloned protein is usually adversely affected by the fused peptide derived from the vector. Therefore the polyclonal sites of the bacteriophage expression vector are suitably located at 30 the N-terminus of lacZ peptides such as in ^ZAPII. It will be appreciated from the foregoing that if an expression vector is utilised as described above the chances of obtaining a 93/25671 PCT / AU93/00294 7 biologically functional enzyme is greatly increased. Isolation of many enzymatically functional xyianase clones in the present invention as described hereinafter has proved the efficiency of this approach. To our knowledge this is the first record of isolation of xyianase cDNA clones 5 with functional enzyme activity from anaerobic fungi based upon the expression of recombinant bacteriophage in E. coli using an expression vector such as that described above. AZAP and AZAP II are examples of such expression vectors. Therefore the term "vectors of similar properties" to AZAP or 10 AZAPII includes within its scope expression vectors having the abovementioned features (i), (ii), (iii) and (iv). It is also clear from the product summary which accompanies the AZAPW vector as supplied by the manufacturer that in relation to fusion protein expression that such fusion proteins may only be screened with 15 antibody probes. Clearly there was no contemplation that the ^ZAPII vector could be utilised for screening of clones involving enzymic expression on a suitable substrate or any direct screening by biological activity. When it is realised that the present invention involves expression in a bacterial host cell such as E. coli of a cDNA of eucaryotic 20 origin (ie. fungal origin) then the novelty of the present invention is emphasised. The screening of xyianase positive recombinant clones may be carried out by any suitable technique based on hydrolysis of xylan. In this procedure the clones may be grown on culture media incorporating 25 xylan and hydrolysis may be detected by the presence of xylanase-positive plaques suitably assisted by a suitable colour indicator. Xyianase positive recombinant clones may then be purified and the cDNA insert in the clones may then be excised into pBluescript (SK(-)) to provide an expression vector of simplified structure when compared 30 to the AZAP II construct which will enhance expression of the xyianase in E. coli. WO 93/25671 PCT/AU93/00294 8 Any suitable E. coli promoter may be used in the expression vector described above. Suitable promoters include lacZ, Tac, Bacteriophage T7 and lambda-PL. The recombinant xyianase enzyme may then be characterised and 5 principal features that have been ascertained are as follows: (i) the cloned xylanases have high specific activity. (ii) the enzyme has no residual activity against cellulose, while many other xylanases possess some cellulase activity. This property of the xyianase is particularly useful in its application to pulp and 10 paper industry to remove xylan and dissociate lignin from plant fibre without damaging cellulose fibre. The high specific activity of the cloned xylanases is an excellent intrinsic property of this fungal xylanases. The expression level of the present constructs of xyianase cDNAs can be further improved by 15 manipulating the gene and promoters. DESCRIPTION OF THE PREFERRED EMBODIMENTS Experimental Methods 1. Microbial strains, vectors and culture media. The anaerobic fungus Neoca/limastix patriciarum (type species) 20 was isolated from a sheep rumen by Orpin and Munn (1986) in Trans. Br. Mycol. Soc. 8§ 178-181 and cultivated in the laboratory for many years under selection by lignocellulose substrates. Host strains for cDNA cloning and characterisation of the recombinant xylanases were E. coli PLK-F, XL 1-Blue and JM83. 25 The vectors were ^ZAPII, pBluescript SK(-) (Stratagene). N. patriciarum culture was maintained in a medium containing 10% rumen fluid as described by Kemp et. al. (1984). E. coli strains were grown in L-broth as described by Sambrook et. al. (1989) for general purposes. The recombinant phage grown in E. coli strains using NZY medium 30 according to Stratagene's instructions. ^WO 93/25671 PCT/AU93/00294 9 2. General recombinant DNA techniques. Agarose-gel electrophoresis, transformation of E. coli and modification of DNA using restriction enzymes and T4 DNA ligase were as described in Sambrook et. al. above. The alkaline lysis method of 5 Birnboim and Doly as described in Nucl. Acids Res. 7 1513-1523 (1976) as employed to isolate plasmid. In vitro DNA amplification by polymerase chain reaction (PCR) was based on the procedure described by Saiki (1989) in PCR Technology (H.A. Erlich, ed) pp. 7-16, M. Stockton Press, New York. 10 3. Cultivation of rumen anaerobic fungus. N. patriciarum for preparation of RNA. N. patriciarum was grown in a rumen fluid-containing medium as described in Kemp et. al. J. Gen. Microbiol. 130 27-37 (1984) in the present of 1 % Avicel at 39 °C and under anaerobic conditions for 48hr 15 (Alternative culture media, such as described by Philips and Gordon in Appln. Environ. Microbiol. |55 1 695-1702 in 1989 and Lowe et. al. in J. Gen. Microbiol. 131 2225-2229 in 1985 can be used). 4 Total RNA isolation. The frozen mycelia were ground to fine powder under liquid nitrogen 20 with a mortar and pestle. 5-10 vol of guanidinium thiocyanate solution (4M guanidinium thiocyanate, 0.5% sodium laurylsarcosine, 25mM sodium citrate, pH7.0, ImM EDTA and 0.1 M B-mercaptoethanol) was added to the frozen mycelia powder and the mixture was homogenised for 5 min with a mortar and pestle and for further 2 min at full speed 25 using a Polytron homogeniser. Total RNA was isolated from the homogenate by ultracentrifugation through a CsCI cushion (Sambrook et. al., 1989). (An alternative method for preparation of total fungal RNA, such as adaptation of the procedure described by Puissant and Houdebine in Bio-Techniques 148-149 in 1990 can be used). 30 5. Polv A* mRNA purification. Poly A+ was purified from the total RNA by Oligo (dT) cellulose chromatography (Sambrook et. al., 1989). ^WO 93/25671 PCT/AU93/00294 10 6. Construction of a cDNA expression library of N. patriciarum. The cDNA library was constructed, using Stratagene's/IZAP cDNA synthesis Kit, basically according to the manufacturer's instructions. The procedure is described briefly as follows: PolyA+ , RNA was 5 converted to the first strand cDNA by reverse transcriptase, using Xhol linker - oligo (dT) primer and 5-methy! dCTP. Double-stranded cDNA was synthesised from the first-strand cDNA by the action of RNase H and DNA polymerase I. After blunting cDNA ends, the cDNA was ligated with EcoR I adaptor, phosphorylated and digested with Xhol to create 10 cDNA with the EcoR I site at 5' region and the Xhol site at 3' region. The cDNA was size-fractionated by 1 % low-melting point agarose gel electrophoresis and 1.2-8Kb sizes of the cDNA were recovered by phenol extraction (Sambrook et. at., 1989). The size-fractionated cDNA was then ligated to the EcoRI/Xhol digested /iZAPII vector. 15 The cDNA library was packaged in vitro and amplified using E. coli PLK-F' as plating cells. 7. Screening xvlanase-oositive recombinant bacteriophage clones. Recombinant phage were grown in E. coli XL 1-Blue in 0.7% top agar containing 0.1% xylan and 10mM isopropyl-f? thio-20 galactopyranoside (IPTG, an inducer for LacZ promoter controlled gene expression). After overnight incubation at 37°C, 0.5% Congo red solution was added over the top agar. After incubation at RT for 15 min, the unbound dye was removed by washing with 1 M NaCI. Xylanase-producing phage plaques were surrounded by yellow haloes 25 against a red background. The xylanase-positive recombinant phage were purified to homogeneity by replating and rescreening the phage as above for 2-3 times. The cDNA insert in xylanase-positive phage were excised into 30 pBluescript SK (-) using R408 helper phage. WO 93/25671 PCT/AU93/00294 11 8. Xvlanase and related-enzvme assays. The cloned enzyme extracts from E. coli harbouring xylanase- positive recombinant plasmids were prepared by harvesting the cells by centrifugation. The cell pellet was suspended in 25mM Tris-CI/ 2mM 5 EDTA containing lysozyme (0.25mg/ml) and incubated on ice for 60 mins. After freezing, thawing and homogenisation, the crude cell lysate was used for enzyme assays. The enzymes were assayed for hydrolysis of xylan or other substrates at 40°C in 50 mM Na-citrate, pH 6.5, except where 10 otherwise indicated in the text. The reducing sugars released from xylan or other plant polysaccharides (Avicel) were measured as described by Lever in Anal. Biochem. 47 273-279 in 1972. Xyianase activity on Kraft pulp was conducted as follows: Kraft pulp was suspended in tap water, and pH was adjusted to pH 7 with 1M 15 H2S04. The xyianase extract was added to the Kraft pulp suspension and the reducing sugar released was measured as above. 9. DNA sequencing. Single-stranded plasmid DNA was prepared basically according to Stratagene's protocol. Sequencing of the resultant DNA was based on 20 the protocol recommended by the manufacturer of the T7 DNA polymerase sequencing kit (Promega). 10. Optimisation of growth conditions of pNX-Tac clone. E.coli strain JM83 harbouring pNX-Tac plasmid grew in LB/Amp(10Qw/ml) at 30°C overnight. One millilitre of the overnight culture 25 was inoculated into 100ml of media as specified in Table 5. IPTG was added at different times of growth. The cultures were grown at 30°C for 17hr, 24hr and 30hr. The cells were harvested for measurement of xyianase yield. Results and discussion 30 Isolation and partial characterisation of xvlanase cDNA clones. A cDNA library consisting of 106 clones was constructed using mRNA isolated from N. patriciarum cells grown with Avicel as sole WO 93/25671 PCT/AU93/00294 12 carbon source. Thirty-one recombinant bacteriophage, which hydrolysed xylan, were identified after an initial screening of 5 x 104 clones from the library and 16 strongly xylanase-positive phage and two weakly xylanase-positive phage were isolated and purified. Xyianase activity of 5 these recombinant bacteriophage clones was initially analysed by scoring xylan-hydrolysis zones (Fig. 1 and Table 1). These 16 strongly xyianase positive clones were originally forwarded to Dr H J Gilbert and Dr G P Hazlewood of The University of Newcastle-upon-Tyne and the AFRC Institute of Animal Physiology and 10 Genetics Research in the United Kingdom who carried out further analysis of these clones which included restriction mapping and hybridization analysis as well as sequencing of the longest clone. In this regard reference should be made to the publication "Homologous catalytic domains in a rumen fungal xyianase: evidence for gene 15 duplication and prokaryolic origin" by H J Gilbert, G P Hazlewood, J I Laurie, C G Orpin and G P Xue which is published in Molecular Microbiology (1992) 6(15) 2065-2072. The longest clone referred to in this reference is designated pNX1 and this corresponds to clone pNPX21 described hereinafter. In the Gilbert et. al. reference described 20 above other plasmids pNX2, pNX3, pNX4, pNX5, pNX6 and pNX7 were produced as a result of truncation of pNXI by restriction enzymes. The clone corresponding to clone pNX1 in E. coli strain XL 1-Blue described above has now been deposited at the International Depository ie. Australian Government Analytical Laboratories on June 22, 1992 25 under accession number N92/27542. In an attempt to obtain more highly active xyianase clones, further screening of 4 x 105 clones from the library was conducted, which resulted in > 200 xylanase-positive clones. Ten highly active clones were isolated and purified. Two of these recombinant bacteriophage 30 clones MNPX29 and /INPX30) have much stronger xyianase activity than previously isolated high activity clones (see Table 1). WO 93/25671 PCT/AU93/00294 13 The cDNA inserts encoding Neocallimastix patriciarum xylanases were in vivo excised from bacteriophage MZAP11) form into plasmid pBluescript SK" form. Several clones with high xyianase activity were analysed for substrate specificity (four clones presented in Table 2). The 5 xylanases produced by these clones have no activity on carboxymethyl-cellulose (CMC, a substrate for endo-glucanase) or Avicel (Avicel is crystalline cellulose and is a substrate for exo-glucanase). The restriction maps of the representative clones are presented in Fig. 2. It appears that these four xyianase cDNAs have the same restriction pattern but 10 differ in length. pNPX13 and pNPX29 have shorter lengths than pNPX21 but they have much higher activity than pNPX21. Interestingly, pNPX30 has a similar length to pNPX21 but it has about 15-fold higher xyianase activity than pNPX21. Because of the remarkable difference in enzyme activity between pNPX21 and pNPX30, the xyianase cDNA 15 of pNPX30 clone was sequenced. The result shows that DNA sequence of pNPX30 shares the same sequence with pNPX21 in a large part of cDNA, but differ in both the 5' and 3' regions. (Fig. 3). pNPX30 cDNA is not full-length. Interestingly, the N-terminus of pNPX30 xyianase has six repeated arginine/glutamic acid residues (Fig. 4). 20 The pH and temperature optima of xylanases produced by pNPX21 and pNPX30 were investigated. These enzymes were active in a wide range of pH and preferably at pH 5 - 8. The thermostability of these enzymes was tested at temperatures from 30°C - 60°C. The enzymes are active at 30°C- 55°C and preferably at 40°C - 50°C. 25 Genetic modification of N. patriciarum xvlanase cDNA pNPX30 (and pNPX21) contains two large repeated domains. Three main constructs were produced from pNPX30. 30 pNXD-Tac pNPX30 plasmid (pNPX21 can also be used) was used as a template for in vitro DNA amplification by PCR for construction of pNXD- WO 93/25671 PCT/AU93/00294 # 14 Tac using primer I and primer IV {Fig.5). The amplified DNA was digested with EcoR1 and Hindi 11 and ligated to EcoR1 and Hindi 11 digested pBTac2 (Boehringer) to produce pNXD-Tac. 5 pNXS-Tac pNXD-Tac plasmid was digested with Hindi 11 and blunted by filling-in with Klenow followed by partial digestion with Seal. After fractionation on LMT agarose gel, the 5.3Kb band was recovered from the gel and ligated to produce the pDGXS construct, which has xyianase 10 activity. pDGXS plasmid was used as a template for in vitro DNA amplification for construction of pNXS-Tac using primer I and primer II (Fig.5). The amplified DNA was digested with EcoRI and Hindi 11 and ligated to EcoRI and Hindi 11 digested pBTac2 vector to produce pNXS-Tac. 15 pNX-Tac pNPX30 plasmid (pNPX21 or other xyianase cDNAs listed in Fig.2 can be used) was digested with Rsal and a 709bp fragment as indicated in Fig.5 was isolated after fractionation on agarose gel electrophoresis. 20 The 709 fragment was ligated to Sma1 and Psti digested pUC18 (Psti end was blunted with T4 DNA polymerase). This construct is designated pNXP2 and the xyianase activity of this construct with the right orientation of truncated xyianase cDNA from pNPX30 confirmed that this fragment of the cDNA encodes a caterlytically functional domain. 25 Two oligonucleotide primers, primer III and primer IV, (Fig.5) were then designed for PCR amplification of the pNXP2 xyianase cDNA insert. The PCR amplified fragment was digested with EcoRI and Hindi 11 and ligated to EcoRI and Hindi 11 digested pBTac2 vector to produce pNX-Tac. 30 These constructs are all modified at the N-terminal sequence of the truncated xyianase cDNA and a translational stop codon (TAA) was introduced into the end of the truncated xyianase coding region. The 93/25671 PCT/AU93/00294 15 expression of xyianase was controlled by the Tac promoter (Fig.6) and xylanases in these constructs are synthesised as nonfusion proteins. The modified xyianase cDNA sequence in pNX-Tac is shown in Fig 7. The specific activity of crude xyianase preparations of pNXD-Tac, 5 pNXS-Tac and pNX-Tac clones were 228, 124 and 672 U/mg of total cellular protein of E.coli respectively, measured in 50mM Na-citrate buffer (pH6) and at 50°C (Fig.5). The xyianase synthesised by the clone pNX-Tac was found mainly in the cell pellet, but a small amount of xyianase (about 5%) was released into the culture medium (Table 3). 10 The pNX-Tac xyianase has a temperature optimum at 50°C and retained >80% of the maximum activity from 40°C to 55°C, and 55% of the activity at 60°C (Fig.8). pNX-Tac xyianase has a broad pH range (Fig 9) and is most active at pH5-7.5, 50% at pH8.5 and 20% at pH9.5. The pNX-Tac xyianase has a high activity in the release of reducing 15 sugar from Kraft pulp at 55 °C and in tap water (pH was adjusted to pH7 with H2S04, see Fig.8) and remains active in the hydrolysis of xylan from the pulp at 55°C and pH7 for at least 3hr (Fig. 10) The pNX-Tac xyianase is able to hydrolyse a significant amount of xylan from Eucalypt and Pine Kraft pulps (Table 4). 20 Optimisation of growth conditions pNX-Tac clone. In order to reduce the cost of xyianase production, growth conditions of E. coli strain JM83 harbouring pNX-Tac plasmid were investigated. Table 5 shows that on a laboratory scale pNX-Tac clone 25 preferably grows in LBMG medium at 30°C for 24 hr, which produced 2-fold higher xyianase yield than LBS. IPTG is preferably added at the beginning of the cultivation (Table 6). Xyianase has many industrial applications, such as the pulp and paper industry, food processing, the feed industry and animal production 30 industry. The enzymes produced by these recombinant xyianase clones have no cellulase activity and have the pH and temperature profile (especially the genetically modified xyianase clone, pNX-Tac) fitted to WO 93/25671 PCT/AU93/00294 16 conditions used for the enzymatic pre-treatment of pulp. Therefore it is believed that the xylanases of the present invention are applicable to the paper and pulp industry. Sandoz Products Pty Ltd, in the USA, have conducted practical 5 trials using their product, Cartazyme, which is a fungal xyianase (crude), active at 30°C-55°C, pH 3 to 5, and contains 2 xylanases, and have found that a 25-33% reduction in chlorine is possible using 1U xylanase/g pulp. Also the product is brighter than when chemicals alone are used. Another advantage of the xyianase is that it is specific 10 whereas chemicals can attack the cellulose at low lignin contents, leading to reduced fibre strength and other undesirable physical characteristics. It is therefore clear that xylanases could become more important in pulp bleaching and recombinant ones particularly so because of their specificity and high level of expression. In particular, the pNX-15 Tac xyianase is very active in hydrolysing of xylan from Kraft pulps. It is also believed that the xyianase of the invention could find a valuable application in the sugar industry and in relation to the treatment of bagasse or other products containing xylan for more efficient disposal as well as for the treatment of feedstock to improve nutritional value. 20 The genetically modified xyianase gene can also be used for modification of rumen bacteria to improve plant fibre utilization by ruminants. It therefore will be apparent from the foregoing that the invention includes within its scope not only the recombinant xyianase described above but also xylanases derived from other anaerobic fungi as described 25 above which may be prepared by the methods described herein. The invention also includes within its scope: (i) DNA sequences derived from these xyianase cDNAs (particularly the sequences in pNPX30, pNXD-Tac, pNXS-Tac and pNX-Tac) and DNA sequences capable of hybridising thereto using a 30 standard nucleic acid hybridisation technique as described in Sambrook et. al. (1989); WO 93/25671 PCT/AU93/00294 17 (ii) a DNA construct containing a DNA sequence as in (i) operably linked to regulatory regions capable of directing the expression or over-expression of a polypeptide having xyianase activity in a suitable expression host; 5 (iii) a transformed microbial host capable of the expression or over-expression of the fungal xyianase, harbouring the above mentioned xyianase constructs; (iv) a polypeptide having xyianase activity produced by expression using a microbial host as in (iii); 10 (v) amino acid sequence derived from these xylanases, truncations and modifications therefrom, by one skilled in the art. Plasmid pNX-Tac in E. coli strain JM83 has been deposited at the International Depository ie. Australian Government Analytical Laboratories 17 March 1993 under accession number N93/12211. 15 In summary the cloning method of the invention is based upon obtaining a large number of recombinant xyianase clones with strong xyianase activity from an anaerobic rumen fungus such as N. patriciarum which were functionally expressed in E. coli. This approach for isolation of fungal xyianase or other plant polysaccharide hydrolases such as 20 cellulases has not been documented prior to this invention. The approach used in this invention is very efficient and requires only a single cloning step to obtain biologically functional recombinant xylanases from an anaerobic fungus. Therefore it takes much less time to obtain biologically functional xyianase clones from a fungal source compared to 25 previous approaches for isolation of plant polysaccharide hydrolases from fungi which are described in the prior art discussed above. The term "essentially" as used in the appended claims includes within its scope sequences having 70-100% identity to those sequences shown in Figs. 3, 4, 5 and 7. vvo 93/25671 PCT/ AU93/00294 18 Table 1 Xyianase activity of recombinant Bacteriophage clones on Xylan - plate assay X ylan - clearing zone 5INPX11 L 1NPX12 S XNPX13 L+++ (9mm) XNPX14 L XNPX15 L+ X.NPX16 L XNPX17 S (4mm) XNPX18 L+ XNPX19 L ?cNPX20 L XNPX21 L+ (7mm) XNPX22 L XNPX23 L XNPX24 L XNPX25 l_+ XNPX26 L++ (8.5mm) XNPX27 L XNPX28 L+ 7.MPX29 L+-t-n- (10.5mm) XNPX30 L++++ (10.5mm) L: Large size S: Small size Values in parenthesis is diameter of zone. XNPX11-28 were isolated from initial screening. A.NPX29 and XNPX30 were isolated after further screening of N.patriciarum cDNA library. SUBSTITUTE SHEET PCT/AU93/00294 19 the cloned xylanases from N. patriciarum Specific activity (U/mg protein) Xylan CMC* Crystalline cellulose pNPX13 41.6 0 0 pNPX21 7.8 0 0 pNPX29 73.5 0 0 pNPX30 113 0 0 * Analysed by CMC plate assay. Crude enzyme extracts were used for enzyme assay. The reactions were carried out at 40°C in 50 mM Na-citrate, pH6.5, containing 0.25% xylan or 1% Avicel. WO 93/25671 Table 2 Specific activity of | SUBSTITUTE SHEET WO 93/25671 PCT/AU93/00294 20 Table 3 Specific activity of pNX-Tac xyianase. Cell pellet Substrate U/mg protein U/ml culture Culture supernant U/ml culture Xylan 672 CMC* 0 Crystalline cellulose 0 (Avicel) 726 23 * Analysed on CMC - plate. E.coli strain JM83 harbouring pNX-Tac plasmid was grown in L-broth at -30°C for 17hrs. Xyianase activity was measured in 50mM Na-citrate pH6 containing 0.25% Xylan at 50°C and the reducing sugar released was measured as described in the method. SUBSTITUTE SHEET wo 93/25671 PCT/AU93/00294 21 Table 4 Reducing sugar released from Kraft pulp. mg reducing sugar released/g dry pulp Xyianase Eucalypt pulp Pine pulp ni/g dry pulp OOO lOpJ " 11.9 ~ 6.97 100*i! 28.9 9.53 The crude xyianase extract from pNX-Tac done was incubated with 6%(W/V) pulp suspension in tap water at pH 7.0. The hydrolysis was carried out at 52°C for 3 hours. SUBSTITUTE SHEET WO 93/25671 PCT/AU93/00294 22 Table 5 Optimisation of growth conditions of ELcoli JM83 harbouring pNX-Tac plasmid. IPTG cell mass at 24hr Xyianase yield (Relative activity) (g/Litre) 17hr 24hr 30hr LBS 0.5mM 10 100% 100% LBSG 11 55% 55% LBMG 0.1 mM 22 168% 168% 0.5mM 22 151% 200% 200% 2.5mM 22 190% 190% LBMHG 0.5mM 20 110% 110% E.coJi strain JM83 harbouring pNX-Tac plasmid was grown in the specified media containing 50}ig/ml Amp at 30°C and IPTG was added at the beginning of the cultivation. Composition of Media, per litre. LBS: Bacto-tryptone 10g Bacto-yeast ext. 5g NaCl 10g Sucrose (0.4%) 4g pH 7.2 LBMG: Bacto-tryptone 5g Bacto-yeast ext. 3g NaCl 0.5g Na2HP0,.12H20 15.1g KH2P04 3g NH4CI 1g Casamino acids 5g Sucrose- 6g CaClj (100mM) 1ml MgS04(1M) 2ml Glucose 4g pH 7.2 LBSG: LBS plus 0.4% Glucose LBMHG: LBMG plus glucose increased to 1% by adding an extra 6g glucose. SUBSTITUTE SHEET WO 93/25671 PCT/AU93/00294 23 Table 6 Optimisation of Induction time of pNX-Tac clone. IPTG added at Xyianase yield (relative activity) Ohr 100% 8hr 82% 16hr 40% ELcoli strain JM83 harbouring pNX-Tac plasmid was grown in LBMG containing 50ng/ml Amp and 0.5 mM IPTG at 30°C for 24 hours. WO 93/25671 PCT/AU93/00294 > 24 LEGENDS Figures 1(a), 1(b), 1(c) and 1(d) Xylan-clearing zones of recombinant bacteriophage clones containing xyianase cDNAs for N. patriciarum concerning cloneS/4NPX13, -ANPX17, 5 y^NPX21 and /INPX26 respectively. Figure 2 Restriction maps of the highly active xyianase clones isolated from Neocaliimastix patriciarum cDNA library. Abbreviations for restriction enzymes: 10 B, BstXI; E, EcoRI; H, Hpal; K, Kpnl; P, Pvull; S, Sacl; Sc, Seal; X, Xhol. Figure 3 The DNA sequence of pNPX30 xyianase cDNA. The sequence typed in small letters comes from the pBluescript SK vector. Figure 4 15 The amino acid sequence of pNPX30 xyianase. The amino acid residues underlined come from the N-terminus of LacZ peptide and encoded by polylinker sequence in the pBluescript SK vector. Figure 5 The genetically modified constructs of the xyianase cDNA 20 vector: . pBTac2 primers: PI: 5'-CGGAATTCATG GCT AGC AGA TTA ACC GTC GGT AAT GGT C Pll: 5'-ATACG TAAGC TTAAA CAGTA CCAGT GGAGG TAG WO 93/25671 PCT/AU93/00294 > 25 Pill: 5'-CGGAA TTCAT GGCTA GCAAT GGTAA AAAGT TTACT G PIV: 5'-ATACG TAAGC TTA AC GAGGA GCGGC AGAGG TGG Abbreviations for restriction enzymes: B, BstX I; E, EcoR I; H. Hpa I; K, Kpn I; P, Pvu II; S, Sac I; Sc, Sea I; X, 5 Xho I. Figure 6 pNX-Tac construct Figure 7 The sequence of the modified xyianase cDNA in pNX-Tac 10 Figure 8 Effect of incubation temperature on the activity of pNX-Tac xyianase. Xyianase assays were performed in 50 mM Na-citrate (pH7) and 0.25% (w/v) xylan at the various temperatures for 30 min. Figure 9 15 Effect of pH on the activity of pNX-Tac xyianase. Xyianase assays were performed at 50 C in 50 mM Na-citrate (pH5-7) or 25 mM Tris-CI / 50 mM NaCl (pH7.5-9.5) with 0.25% xylan for 30 min. The pHs of the buffers were measured at room temperature. Figure 10 20 Time course of pNX-Tac xyianase activity on eucalypt Kraft pulp. Hydrolysis was carried out at 55°C in tap-water suspended pulp at pH 7.0. I SUBSTITUTE SHEET WO 93/25671 PCT/AU93/00294 2 ssqsn 26 </div>

Claims

<div class="application article clearfix printTableText" id="claims"> CLAIMS:

1. A method of cloning of xyianase clones from an anaerobic rumen fungus including the steps of: (i) cultivation of an anaerobic rumen fungus; (ii) isolating total RNA from the culture in step (i); (iii) isolating poly A+ mRNA from the total RNA referred to in step (ii); (iv) constructing a cDNA expression library; (v) ligating cDNA to a bacteriophage expression vector selected from AZAP, ^ZAPII or vectors of similar properties; (vi) screening of xyianase positive recombinant clones in a culture medium incorporat'ng xylan by detection of xylan hydrolysis; and (vii) purifying xyianase positive recombinant clones.

2. A method as claimed in claim 1 wherein the expression vector is AZAPU.

3. A method as claimed in claim 1 wherein the detection of enzyme hydrolysis is carried out using a colour indicator Congo red.

4. A method as claimed in claim 1 wherein after production of xyianase positive clones the cDNA insert in such clones were excised into p Bluescript SK(-) using helper phage.

5. A method as claimed in claim 4 wherein the helper phage is R408 helper phage.

6. Xyianase positive recombinant clones produced by the method of claim 1.

7. Xyianase positive recombinant clones having the following properties: (i) production of xylan clearing zones in a culture containing xyianase cDNA derived from N. patriciarum: (ii) having activity in hydrolysis of xylan but having no activity in relation to hydrolysis of CMC or crystalline cellulose. I SUBSTITUTE SHEET I 25 2 9 37 27

8. Recombinant xyianase clone pNPX21 deposited at the' Australian Government Analytical Laboratories on June 22, 1992 under accession number N92/27542.

9. An isolated DNA molecule encoding a truncated xyianase polypeptide, wherein said truncated xyianase polypeptide is encoded by a nucleotide sequence essentially as shown at nucleotide 25 to 2481 in Figure 3.

10. A truncated xyianase polypeptide including an amino acid sequence essentially as shown at amino acid 42 to 644 in Figure 4.

11. An isolated DNA molecule encoding a truncated xyianase polypeptide, wherein said truncated xyianase polypeptide is encoded by a nucleotide sequence essentially corresponding to that of the xylanase-encoding portion of pNXD-Tac shown in Figure 5.

12. An isolated DNA molecule encoding a truncated xyianase polypeptide, wherein said truncated xyianase polypeptide is encoded by a nucleotide sequence essentially corresponding to that of the xylanase-encoding portion of pNXS-Tac shown in Figure 5.

13. An isolated DNA molecule encoding a truncated xyianase polypeptide, wherein said truncated xyianase polypeptide is encoded by a nucleotide sequence essentially corresponding to that of the xylanase-encoding portion of pNX-Tac shown in Figure 5.

14. Primer PI shown in FIG 5.

15. Primer Pll shown in FIG 5.

16. Primer PHI shown in FIG 5.

17. Primer PIV shown in FIG 5.

18. An isolated DNA molecule encoding a truncated xyianase polypeptide, wherein said truncated xyianase is encoded by a nucleotide sequence esseoiialbf corresponding to that shown in Figure 7.

19. Xylanases produced from the recombinant xyianase clones of claim 6.

20. Xylanases produced from the recombinant xyianase clones of claim 7.

21. A DNA construct containing a DNA sequence as claimed in claim 9 operably linked to regulatory regions capable of directing the expression or over-expression of a polypeptide having xyianase activity in a suitable expression host. WO 93/25671 PCT/AU93/00294 28

22. A DNA construct containing a DNA sequence as claimed in claim 11 operably linked to regulatory regions capable of directing the expression or over-expression of a polypeptide having xyianase activity in a suitable expression host.

23. A DNA construct containing a DNA sequence as claimed in claim 12 operably linked to regulatory regions capable of directing the expression or over-expression of a polypeptide having xyianase activity in a suitable expression host.

24. A DNA construct containing a DNA sequence as claimed in claim 13 operably linked to regulatory regions capable of directing the expression or over-expression of a polypeptide having xyianase activity in a suitable expression host.

25. A DNA construct containing a DNA sequence as claimed in claim 18 operably linked to regulatory regions capable of directing the expression or over-expression of a polypeptide having xyianase activity in a suitable expression host.

26. A transformed microbial host capable of the expression or over expression of fungal xyianase harbouring the xyianase construct of claim 21.

27. A transformed microbial host capable of the expression or over expression of fungal xyianase harbouring the xyianase construct of claim 22.

28. A transformed microbial host capable of the expression or over expression of fungal xyianase harbouring the xyianase construct of claim 23.

29. A transformed microbial host capable of the expression or over expression of fungal xyianase harbouring the xyianase construct of claim 24.

30. A transformed microbial host capable of the expression or over expression of fungal xyianase harbouring the xyianase construct of claim 25. SUBSTITUTE SHEET 25 2 9 3 7 29

31. A polypeptide having xyianase activity produced by expression using a microbial host of claim 26.

32. A polypeptide having xyianase activity produced by expression using a microbial host of claim 27.

33. A polypeptide having xyianase activity produced by expression using a microbial host of claim 28.

34. A polypeptide having xyianase activity produced by expression using a microbial host of claim 29.

35. A polypeptide having xyianase activity produced by expression using a microbial host of claim 30.

36. A polypeptide with amino acid sequences derived from the polypeptide of claim 31 including truncated and modified forms thereof.

37. A polypeptide with amino acid sequences derived from the polypeptide of claim 32 including truncated and modified forms thereof.

38. A polypeptide with amino acid sequences derived from the polypeptide of claim 33 including truncated and modified forms thereof.

39. A polypeptide with amino acid sequences derived from the polypeptide of claim 34 including truncated and modified forms thereof.

40. A polypeptide with amino acid sequences derived from the polypeptide of claim 35 including truncated and modified forms thereof.

41. Plasmid pNX-Tac lodged at the Australian Government Analytical Laboratories on March 17, 1993 under accession number N93/12211.

42. An isolated cDNA molecule which encodes a functional Neocallimastix xyianase.

43. An isolated cDNA molecule which encodes a fungjtJjfifii Neocallimastix patriciarum xyianase. L WO 93/25671 PCT/ AU93/00294 25QPRTI 30

44. A transformed microbial host capable of the expression or over expression of fungal xyianase harbouring the cDNA molecule of claim 42. expression of fungal xyianase harbouring the cDNA molecule of claim 43. 46. A polypeptide having xyianase activity produced by expression using the microbial host of claim 44. 47. A polypeptide having xyianase activity produced by expression using the microbial host of claim 45. 48. Amino acid sequences derived from the peptide of claim 46 including truncated and modified forms thereof. 49. Amino acid sequences derived from the peptide of claim 47 including truncated and modified forms thereof.

45. A transformed microbial host capable of the expression or over </div>