AU696724B2 - Recombinant xylanase - Google Patents

Description

OHI

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DAI[ 04/01/94 G;ATE 24/03/94 PCT NUMBER rPGTfAU 1 0?94 1I1iIU I111113111 I 11111111111111ll11111 111 IN l ilt AU9342983

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(51) International Patent Gassification C12N 0 '38, 15/56 (11) International Publication Number:. WO 93/25671 A' j(43) International Publication Date: 23 lievemrber 1993 (23.12.93) (21) International Application Number: (22) International Filing Date: Priority data: PL 2985 17 June I PL 3238 29J1unelI PL8100 I April 1 PCT:,AU93 00294 17 June 1993 (17.06.93) 992 (17.06.92) 992 (29.06.92) '93 (01.04.93) (71) Applicant(for all designated Stotes ex'cept V&t COMMON- WEALTH SCIENTIFIC AND) INDUSTRIAL RE- SEARCH ORGANISATION IAU/AUI; Limestone Avenue, Campbell, Canberra, ACT 2601 (AU).

(72) Inventor; and Irnentor/Applicant (for US onloy: XUE, Gang, Ping IAU/ AU]; 20 Camira Street, St.Lucia, Brisbane, QLI) 4067

(AUJ).

o'f G, KeY\c-!*joA Tprrace, N\etacc6*k.- Upf-rY0 ~MCI ?MRML4T 0 e (74) Agent,. KELLY. Robin, Thomas; Fisher Kelly. Level One, 349 Coronation Drive. Milton, QLI) 4064 (AV), (81) Designated States: AT, AU, 131, 13O, BR, BY, CA, CHI, CZ. DE. DY, ES, Fl, G13, HU, JP. KP, KR, KZ, LK, LL, MG, MN. MNVI NL, NO, NZ, PL, PT, RO, R U, SI). SE, SY, UA. 'US, VN, European patent (AT, BE, CH, DE, ES, FR, GB. GR. IE, IT, LU. MC, NL, PT, SE), OAPI patent (BF. Iii, CF. CG, Cl. CM, GA.

ON, MIL, MR, NE, SN, TD, TO).

Published Ifith internationial search report, Before the expira;ion of the :itne limnit for amending the claims and to be .republished in the ere;nz of the receipt of amendments.

Lk4L CI r~ET Vaf o (54) Title: RECOMBINANT XYLANASE (57) Abstract A method of cloning of xylanase clones fromn an anaerobic rumen fungus including the steps or: cultivation of an anaer, obic rumen fungus;, (ii) isolating total RNA from the culture in step (iii) isolating poly A+ mnRNA from the total RNA referred to in step (iv) constructing a cDNA expression library;, ligating cDNA to a bacteriophage expression vector selected from ?ZAP, ?ZAPII or vectors of similar properties; (vi) screening of xylanase positive recombinant clones in a culture medium incorporating xylan by detection of xylan hydrolysis; and (Vi) purifying xylanase positive recombinant clones. There is also provided xylanase positive recombinant clnecs produced by the above-mentioned method as well as xylanase positive recombinant clones having the following properties: production of xylan clearing zones in a culture containing xylanase 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. There is also provided various cDNA molecules which may be utilised in the above-mentioned method, WO 93/25671 PCIYA1J93100294 1

TITLE

"RECOMBINANT XYLANASE" FIELD OF INVENTION This invention relates to a recombinant xylanase derived from an anaerobic fungus and a method of production of the recombinant xylanase and clones utilised in the method.

BACKGROUND ART Xylan is a maior component of hemicellulose and the second major component of plant fibre. Xylan consists of a backbone of P-1,4-linked xylose units. The enzymic cleavage of B-1,4-xylosidic linkages is performed by endo-B-1,4-xylanases (xylanases). Many microorganisms produce extracellular xylanases. In the past decade, many xylanase genes were isolated from lignocellulolytic bacteria, but isolation of xylanase genes from fungi with functional expression in E. coli has not been documented prior to this invention.

Lignocellulolytic fungi usually produce more active xylanase than bacteria, in particular, the anaerobic fungus Neocallimastix patriciarum, isolated from the sheep rumen, has a high capacity for xylan degradation.

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: Reymond et. al. Gene 110 (1992) 57-63; (ii) Wong et. al. Clin. Reviews in Biotechnology 12 413-435 (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); Joblin et. al. FEMS Microbiology Letters 65 (1989) 119- 122; WO 93/256711 I'Cf/AU93/00294 2 (vi) Lowe et. Applied and Environmental Microbiology June 1987 pp 1210-1215; and (vii) Lowe et. al. Applied and Environmental Microbiology June 1987 pp 1216-1223.

Cloning of xylanase genes from bacteria can be achieved by isolation of enzymatically active clones from genomic libraries established in E. coli. However this approach for isolation of xylanase genes from fungal genomic libraries with functional expression of xylanase is not possible. This is because fungi are eucaryotic 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.

The cDNA cloning approach can be used to overcome the posttranscriptional 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 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. coli are 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 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 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 PC/AlU93/00294 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 24 411-422 (1988); Morosoli and Durand FEMS Microbiology Letters 51 217-224 (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 xylanase is also required. It usually takes more than one year to obtain a functional enzyme clone using the above approaches.

Isolation of fungal xylanase 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 enzymatically functional xylanase 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 xylanase clones by the convenient xylan-agar plate technique is difficult. Bacteriophage 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, Agtl 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 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 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 93/25671f PCI/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.

In relation to isolation of a fungal xylanase gene, the only report that exists so far prior to this invention is the abovementioned Morosoli and Durand reference which describes isolation of a xylanase gene from yeast Cryptococcus albidus using differential hybridization techniques.

However, this reference does not describe the production of biologically functional enzymes from this xylanase gene.

BROAD STATEMENT OF INVENTION It is an object of the invention to provide a recombinant xylanase from an anaerobic rumen fungus which may be of use commercially in relation to hydrolysis of xylan.

A further object of the invention is to provide a method of cloning of xylanase cDNAs from an anaerobic rumen fungus which may encode the recombinant xylanase of the invention.

A further object of the invention is to provide xylanase clones which may be produced in the abovementioned method.

The method of cloning of the invention includes the following steps: (i) (ii) (iii) (iv) (v) (vi) cultivation of an anaerobic rumen fungus; isolating total RNA from the culture in step isolating poly A' mRNA from the total RNA referred to in step (ii); constructing a cDNA expression library; ligating cDNAs to a bacteriophage expression vector selected from AZAP, AZAP II or vectors of similar properties; screening of xylanase positive recombinant clones in a culture medium incorporating xylan by detection of xylan hydrolysis; and WO 93/2571 PCIAU93/00294 (vii) purifying xylanase positive recombinant clones.

In step above in relation to preparation of the recombinant xylanase, from anaerobic fungi, particularly alimentary tract fungi, may be cultivated as described hereinbelow. These fungi are strict anaerobes and may be exemplified by Neocallimastix patriciarum, Neocallimastix frontalis, Neocallimastix hurleyensis, Neocallimastix stanthorpensis, Sphaeromonas communis, Caecomyces equi, Piromyces communis, Piromyces equi, Piromyces dumbonica, Piromyces lethargicus, Piromyces mai, Ruminomyces elegans, Anaeromyces mucronatus, Orpinomyces 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.

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 filtration and subsequently lysed in appropriate cell lysis buffer by mechanical disruption. A suitable RNA preserving compound may also he 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 technique such as by ultracentrifugation through a CsCl 2 cushion or alternative technique as described by Sambrook et. al. in Molecular Cloning; A Laboratory Manual 2 1 .d 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 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 93/256717 'PC1/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 mRNA may then be isolated from the total RNA by affinity chromatography on a compound containing multiple thymine residues such as oligo (dT) cellulose, A'ternatively a compound containing multiple uracil residues may be used such as poly Sephadex. The poly mRNA may then be eluted from the affinity column by a suitable buffer.

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 may subsequently be fractionated to a suitable size and may be ligated to the bacteriophage expression vector, preferably AZAP 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 is important in that such expression vector should include the following features: having an E. coli promoter; (ii) having a translation start codon; (iii) having a ribosomal binding site; (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 the N-terminus of lacZ peptides such as in AZAPII.

It will be appreciated from the foregoing that if an expression vector is utilised as described above the chances of obtaining a WO 93/25671 PCI/AU93/00294 7 biologically functional enzyme is greatly increased, Isolation of many enzymatically functional xylanase 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 xylanase cDNA clones 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 AZAPII includes within its scope expression vectors having the abovementioned features (iii) and (iv).

It is also clear from the product summary which accompanies the AZAPII vector as supplied by the manufacturer that in relation to fusion protein expression that such fusion proteins may only be screened with antibody probes. Clearly there was no contemplation that the AZAPII 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 origin (ie. fungal origin) then the novelty of the present invention is emphasised.

The screening of xylanase 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 xylan and hydrolysis may be detected by the presence of xylanasepositive plaques suitably assisted by a suitable colour indicator.

Xylanase positive recombinant clones may then be purified and the cDNA insert in the clones may then be excised into pBluescript to provide an expression vector of simplified structure when compared to the AZAP II construct which will enhance expression of the xylanase in E. coli.

WO 93/25671 WI('/A93/9OOZ94 8 Any suitable E. coli promoter may be used in the expression vector described above. Suitable promoters include lacZ, Tac, Bacteriophage T, and lambda-P

L

The recombinant xylanase enzyme may then be characterised and principal features that have been ascertained are as follows: 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 xylanase is particularly useful in its application to pulp and 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 xylanase cDNAs can be further improved by manipulating the gene and promoters.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Experimental Methods 1. Microbial strains, vectors and culture media.

The anaerobic fungus Neocallimastix patriciarum (type species) was isolated from a sheep rumen by Orpin and Munn (1986) in Trans.

Br. Mycol. Soc. 86 178-181 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, XL1-Blue and JM83.

The vectors were AZAPII, pBluescript (Stratagene). N.

patriciarum cul+jire 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 according to Stratagene's instructions.

WO 9)3/2$71 WO 9/2561 I*1 1 /7AV93/00294 9 2, General recombinant DNA techniquas.

Agarose-gel electrophoresis, transformation of E. co/i and modification of DNA using restriction enzymes and T4 DNA ligase were as described in Sambrook et. al. above. The alkaline lysis method of Birnboim and Doly as described in Nucl. Acids Res. 7 1513-1523 (1976) as employed to isolate plasmid. In vitro DNA amplification by po' merase chain reaction (PCR) was based on the procedure described by Saiki (1989) in PCR Technology Erlich, ed) pp. 7-16, M.

Stockton Press, New York.

3. Cultivation of rumen anaerobic fungus, N. patriciarum for preparation of RNA.

N. patriciarum was grown in a rumen fluid-L.,,vitaining 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 (Alternative culture media, such as described by Philips and Gordon in Appln. Environ. Mic;obiol. 55 1695-1702 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 with a mortar and pestle. 5-10 vol of guanidinium thiocyanate solution (4M guanidinium thiocyanate, 0.5% sodium laurylsarcosine, sodium citrate, pH7.0, 1mM 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 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).

5. Poly A' mRNA purification.

Poly A' was purified from the total RNA by Oligo (dT) cellulose chromatography (Sambrook et. al., 1989).

WO 93/25671 PCI/AU93/00294 S Construction of acDNA prssion library of N. mariciaru The cDNA library was cons, ructed, using Stratagene's AZAP cDNA synthesis Kit, basically according to the manufacturer's instructions.

The procedure is described briefly as follows: PoiyA RNA was converted to the first strand cDNA by reverse transcriptase, using Xhol linker oligo (dT) primer and 5-mothyl 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 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 eDNA was then ligated to the EcoRI/Xhol digested AZAPII vector.

The cDNA library waS packaged in vitro and amplified using E. coli PLK-F' as plating cells.

7. Screening xayl _ase-ositLv_ re om an bacteriohae cones.

Recombinant phage were grown in E. co// XL1-Blue in 0.7% top agar containing 0.1% xylan and 10mM isoprorF/l--thiogalactopyranoside (IPTG, an inducer for LacZ promoter controlled genp expression). After overnight incubation at 37°C, 0,5% Congo rt, solution was added over the top agar. After incubation at RT for min, the unbound dye was removed by washing with 1 M NaCI.

Xylanase-producing phage plaques were surrounded by yellow haloes against a red background.

The xylanase-positlve 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 pBluescript SK using R408 helper phage, WO 93/25671 PC1TAU93/00294 11 8. Xyvanase and related-enzyme assays.

The cloned enzyme extracts from E. coli harbouring xylanasepositive recombinant plasmids were prepared by harvesting the cells by centrifugation. The cell pellet was suspended in 25mM Tris-CI/ 2mM EDTA containing lysozyme (0.25mg/ml) and incubated on ice for 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 0 C in 50 mM Na-citrate, pH 6.5, except where 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.

Xylanase activity on Kraft pulp was conducted as follows: Kraft pulp was suspended in tap water, and pH was adjusted to pH 7 with 1 M HzSO4. The xylanase 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 the protocol recommended by the manufacturer of the T7 DNA polymerase sequencing kit (Promega).

Optimisation of growth conditions of pNX-Tac clone.

E.coli strain JM83 harbouring pNX-Tac plasmid grew in LB/Ampioo,iml at 300C overnight. One millilitre of the overnight culture was inoculated into 100ml of media as specified in Table 5. IPTG was added at different times of growth. The cultures were grown at 300C for 17hr, 24hr and 30hr. The cells were harvested for measurement of xylanase yield.

Results and discussion 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 NVO 9/25671 PC/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. Xylanase activity of these recombinant bacteriophage clones was initially analysed by scoring xyla;-hydrolysis zones (Fig. 1 and Table 1).

These 16 strongly xylanase 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 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 xylanase: evidence for gene 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 above other plasmids pNX2, pNX3, pNX4, pNX5, pNX6 and pNX7 were produced as a result of truncation of pNX1 by restriction enzymes.

The clone corresponding to clone pNX1 in E. coli strain XL1-Blue described above has now been deposited at the International Depository ie. Australian Government Analytical Laboratories on June 22, 1992 under accession number N92/27542.

In an attempt to obtain more highly active xylanase clones, further screening of 4 x 10 s 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 clones (ANPX29 and ANPX30) have much stronger xylanase activity than previously isolated high activity clones (see Tab!q 1).

WO 93/25671 I'Cr/AU93/00294 13 The cDNA inserts encoding Neocallimastix patriciarum xylanases were in vivo excised from bacteriophage (AZAP11) form into plasmid pBluescript SK' form. Several clones with high xylanase activity were analysed for substrate specificity (four clones presented in Table The xylanases produced by these clones have no activity on carboxymethylcellulose (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 xylanase cDNAs have the same restriction pattern but differ in length. pNPX13 and pNPX29 have shorter lengths than pNPX21 but they have much higher activity than pNPX21. Interestingly, has a similar length to pNPX21 but it has about 15-fold higher xylanase activity than pNPX21. Because of the remarkable difference in enzyme activity between pNPX21 and pNPX30, the xylanase cDNA 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. pNPX30 cDNA is not full-length. Interestingly, the N-terminus of pNPX30 xylanase has six repeated arginine/glutamic acid residues (Fig. 4).

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 300C 600C. The enzymes are active at 30°C- 55°C and preferably at 40 0 C 500C.

Genetic modification of N. patriciarum xvlanase cDNA (and pNPX21) contains two large repeated domains.

Three main constructs were produced from pNXD-Tac 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 PCI'/AU93/00294 14 Tac using primer I and primer IV (Fig.5). The amplified DNA was digested with EcoR1 and Hind 11 and ligated to EcoR1 and Hind 11 digested pBTac2 (Boehringer) to produce pNXD-Tac.

pNXS-Tac pNXD-Tac plasmid was digested with Hind 111 and blunted by filling-in with Klenow followed by partial digestion with Scal. After fractionation on LMT agarose gel, the 5.3Kb band was recovered from the gel and ligated to produce the pDGXS construct, which has xylanase activity. pDGXS plasmid was used as a template for in vitro DNA amplification for construction of pNXS-Tac using primer I and primer II The amplified DNA was digested with EcoR1 and Hindi 11 and ligated to EcoR1 and Hind 111 digested pBTac2 vector to produce pNXS- Tac.

pNX-Tac plasmid (pNPX21 or other xylanase 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.

The 709 fragment was ligated to Smal and Pstl digested pUC18 (Pstl end was blunted with T4 DNA polymerase). This construct is designated pNXP2 and the xylanase activity of this construct with the right orientation of truncated xylanase cDNA from pNPX30 confirmed that this fragment of the cDNA encodes a caterlytically functional domain.

Two oligonucleotide primers, primer III and primer IV, (Fig.5) were then designed for PCR amplification of the pNXP2 xylanase cDNA insert. The PCR amplified fragment was digested with EcoR1 and Hind111 and ligated to EcoR1 and Hind 111 digested pBTac2 vector to produce pNX- Tac.

These constructs are all modified at the N-terminal sequence of the truncated xylanase cDNA and a translational stop codon (TAA) was introduced into the end of the truncated xylanase coding region. The WO 93/25671 2PCIT/A93/00294 expression of xylanase was controlled by the Tac promoter (Fig.6) and xylanases in these constructs are synthesised as nonfusion proteins. The modified xylanase cDNA sequence in pNX-Tac is shown in Fig 7.

The specific activity of crude xylanase preparations of pNXD-Tac, pNXS-Tac and pNX-Tac clones were 228, 124 and 672 U/mg of total cellular protein of E.ccli respectively, measured in 50mM Na-citrate buffer (pH6) and at 500C (Fig.5). The xylanase synthesised by the clone pNX-Tac was found mainly in the cell pellet, but a small amount of xylanase (about was released into the culture medium (Table 3).

The pNX-Tac xylanase has a temperature optimum at 50°C and retained of the maximum activity from 40°C to 55CC, and 55% of the activity at 60 0 C (Fig.8). pNX-Tac xylanase has a broad pH range (Fig 9) and is most active at pH5-7.5, 50% at pH8.5 and 20% at The pNX-Tac xylanase has a high activity in the release of reducing sugar from Kraft pulp at 55 0 C and in tap water (pH was adjusted to pH7 with H 2

SO

4 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 xylanase is able to hydrolyse a significant amount of xylan from Eucalypt and Pine Kraft pulps (Table 4).

Optimisation of growth conditions pNX-Tac clone.

In order to reduce the cost of xylanase 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 preferably grows in LBMG medium at 300C for 24 hr, which produced 2-fold higher xylanase yield than LBS. IPTG is preferably added at the beginning of the cultivation (Table 6).

Xylanase has many industrial applications, such as the pulp and paper industry, food processing, the feed industry and animal production industry. The enzymes produced by these recombinant xylanase clones have no celiulase activity and have the pH and temperature profile (especially the genetically modified xylanase clone, pNX-Tac) fitted to WO 93/25671 W /1'1AU93/00294 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 trials using their product, Cartazyme, which is a fungal xylanase (crude), active at 30 0 C-55 0 C, pH 3 to 5, and contains 2 xyianases, 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 xylanase is that it is specific 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- Tac xylanase is very active in hydrolysing of xylan from Kraft pulps.

It is also believed that the xylanase 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.

The genetically modified xylanase gene can also be used for modification of rumen bacteria to improve plant fibre utilization 6y ruminants.

It therefore will be apparent from the foregoing that the invention includes within its scope not only the recombinant xylanase described above but also xylanases derived from other anaerobic fungi as described above which may be prepared by the methods described herein.

The invention also includes within its scope: DNA sequences derived from these xylanase cDNAs (particularly the sequences in pNPX30, pNXD-Tac, pNXS-Tac and pNX-Tac) and DNA sequences capable of hybridising thereto using a standard nucleic acid hybridisation technique as described in Sambrook et. al. (1989); WO 93/25671 PCT/A93/,, 17 (ii) a DNA construct containing a DNA sequence as in operably linked to regulatory regions capable of directing the expression or over-expression of a polypeptide having xylanase activity in a suitable expression host; (iii) a transformed microbial host capable of the expression or overexpression of the fungal xylanase, harbouring the above mentioned xylanase constructs; (iv) a polypeptide having xylanase activity produced by expression using a microbial host as in (iii); 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.

In summary the cloning method of the invention is based upon obtaining a large number of recombinant xylanase clones with strong xylanase activity from an anaerobic rumen fungus such as N. patriciarum which were functionally expressed in E. coli. This approach for isolation of fungal xylanase or other plant polysaccharide hydrolases such as 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 'sss time to obtain biologically functional xylanase clones from a fungal source compared to 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.

WO 93/25671 PCI/A U93/00294 18 Table 1 Xytanase activity of recombinant Bacteriophage clones on Xylan plate assay Xylan clearing zone XNPXI I XNPX1 2 XNPX1 3 XNPX14 ;XNPXI 6 XN'PX17 XNPX18 XNPX19 XLNPX21 XNPX22 2LNPX23 )XNPX24 XNPX26 XNPX27 XNPX28 XNPX29

L

S

(9mm)

L

L-

L

S (4mm)

L+

L

L

L

L

L+

L

105m (1 L: Large size S: Small size Values in parenthesis is diameter of zone.

XNPXI 1-28 were isolated from initial screening.

XLNPX29 and M~PX30 were isolated after further screening of NUpatriclawum oDNA library.

S0TITUTE SHET 93/25671 KI(V193/Of,294 19 Table 2 Specific activity of the cloned xylanases from N. patriciarum Specific activity (U/mg protein) Xytan CMC* Crystalline cellulose pNPX13 41.6 0 0 pNPX21 7.8 0 0 pNPX29 73.5 0 0 113 0 0 *Analysed by CIVC plate assay.

Crude enzyme extracts were used for enzyme, assay. The reactions were carried out at 40 0 C in 50 mnM Na-citrate, pH6.5, contaiNing 0.25% xylan or 1% AVicel.

SUBSTITUTE SHEET7 WO 93/25671 110/A U93/0029,i Table 3 Specific activity of pNX-Tac xylanase.

Cell elletCulture supernant Substrate U/mg protein U/mi culture U/mi culture Xylan 672 726 23 CMC* 0 Crystalline cellulose 0 (Avicel) *Analysed on CMC plate.

Ecoli strain JM83 harbouring pNX-Tac plasmid was grown in L-broth at ,3000 for M~rs.

Xylanase activity was measured in 50mM Na-citrate pH-6 containing 0.25% Xylan at 5000 and the ieducing sugar released was measured as described in the method.

SUBSTITUTE SHEET WO 93/26671 KWCUA U93/002 94 21 Table 4 Reducing sugiar released from Kraft pulp.

mg reducing sugar releasecllg dry pulp Xylanase Eucalypt pulp Pine pulp gilg dry pulp 0 0 0 10J11 11.9 6.97 1 OgII 28.9 9.53 The crude xylanase extract from pNX-Tac clone was incubated with 6%(WN) pulp suspension in tap water at pH-17.0. The hydrolysis was carried out at 52*C for 3 houls.

SUBSTITUTE SHEET WVO 93/25671 I 'C1I/Ai93/0O29,1 Table Optimisation of growth conditions of Ecoll JM83 harbouring pNX-Tac plasmid.

Xylanase yield IPTG cell mass (Relative activity) at 24hr (gf~itre) 17hr 24hr LBS 0.5mM 10 100% 100% LBSG 11 55% LBMG 0.1mM 22 168% 10-8% 22 151%/ 200% 200% 22 190% 190% LBMHG 0.5mM 20 110% 110% E-cofi strain JM83 harbouring pNX-Tac, plasmid was grown in the specified media containing 50figfiml Amp at 3000 and IPTG was added at the beginning of the cultivation.

Composition of Media, per litre.

LBS: Bacto-tryptone Bacto-yeast ext, NaCI Sucrose pH 7.2 LBMG: Bacto-tryptone Bacto-yeast ext.

NaG! NaHPO 4 .1 2H 2 0

KH

2

POA

NH

4

C(

Casamino acids Sucrose- Gal (100mM) MgSO 4 (iM) Glucose pH 7.2 log 5g 1log LBSG: LBS plus 0.4% Glucose 5g 3g 0.5g 15.1 g 3g I g 6g imi 2m1 4g LBMHG: LBMG plus glucose increased to 1%0/ by adding an-extra 6g glucose.

SUBSTITUTE SHEET WO 93/25671 lbC%/ IM 93/00294 23 Table 6 Optimisation of Induction time of pNX-Tac clone.

I PTG added at Xylanase yield (relative activity) Ohr 100% 8hr 82% l6hr Ecoli strain JM83 harbouring pNX-Tao plasmid was grown in LBMG containing Amp and 0.5 mM 1PTG at 30 0 C for 24 hours.

SUBITUTE s~rEr! WO 93/25671 PC''/A 193/00294 24

LEGENDS

Figures 1 1 1 and 1 (d) Xylan-clearing zones of recombinant bacteriophage clones containing xylanase cDNAs for N. patriciarum concerning clones ANPX1 3, A NPX 17, ANPX21 and ANPX26 respectively.

Figure 2 Restriction maps of the highly active xylanase clones isolated from Neocailimastix patriciarum cDNA library.

Abbreviations for restriction enzymes: 13, BstXl;- E, EcoRI;- H, Hpal; K, Kpnl;- P, Pvull; S, Sacl; Sc, Scal; X, Xhol.

Figure 3 The DNA sequence of pNPX3O xyfanase cDNA. The sequence typed in small letters comes from the pBluescript SIK vector.

Figure 4 The amino acid sequeoce of pNPX3O xylanase. The amino acid residues underlined come from the N-terminus of LacZ peptide and encoded by polylinker sequence in the pBluescript SK vector.

Figure The genetically modified constructs of the xylanase cDNA vector: pBTaC 2 primers: P1: 5'-CGGAATTCATG GCT AGC AGA TTA ACC GTC GGT AAT GGT C Pi1: 5'-ATACG TAAGC TTAAA CAGTA CCAGT GGAGG TAG UBTUTE SHEE7j WO 93/25671 WO 93/2571 I1CTAU93/00294 Pill: 5'-CGGAA TTCAT GGCTA GCAAT OGTAA AAAGT TTACT G PIV: 5-ATACG TAAGC TTAAC GAGGA GCGGC AGAGG TGG Abbreviations for restriction enzymes: 13, BstX 1; E, EcoR 1; Hpa 1; K, Kpn 1; P, Pvu 11; S, Sac 1; Sc, Sca I; X, Xho 1.

Figure 6 pNX-Tac construct Figure 7 The sequence oi the modified xylanase cDNA in pNX-Tac Figure 8 Effect of incubation temperature on the activity of pNX-Tac xylanase.

Xylanase assays were performed in 50 mM Na-citrate (pH- 7 and 0.25% xylan at the various temperatures for W3C min.

Figure 9 Effect of pH on the activity of pNX-Tac xylanase.

Xylanase assays were performed at 50 C in 50 mM Na-citrate (pH5-7) or 25 mM Tris-CI 50 mM NaCI (pH-7.5-9.5) with 0.25% xylan for min. The pHs of the buffers were measured at room temperature.

Figure Time course of pNX-Tac xylanase activity on eucalypt Kraft pulp.

Hydrolysis was carried out at 5500 in tap-water suspended pulp at pH SUB8STITUTE SHEET

Claims (24)

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

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

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 xylanase positive clones the cDNA insert in such clones were excised into p Bluescript using helper phage. A method as claimed in claim 4 wherein the helper phage is R408 helper phage.

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

7. Xylanase positive recombinant clones having the following properties: production of xylan clearing zones in a culture containing xylanase 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. ISUBSTITUTE SHEET

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

9. A truncated xylanase polypeptide including an amino acid sequence essentially as shown at anino acid 42 to 644 in Figure 4. An isolated DNA molecule encoding a truncated xylanase polypeptide, wherein said truncated xylanase polypeptide is encoded by a nucleotide sequence essentially corresponding to that of the xylanase-encoding portion of pNXD-Tac shown in Figure

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

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

13. Primer PI shown in Figure

14. Primer PII shown in Figure S 20 15. Primer PII shown in Figure S16. Primer PIV shown in Figure

17. An isolated DNA molecule encoding a truncated xylanase polypeptide, wherein said truncated xylanase is encoded by a nucleotide sequence :essentially corresponding to that shown in Figure 7. 25 18. Xylanases produced from the recombinant xylanase clones of claim 6. 19, Xylanases produced from the recombinant xylanase clones of claim 7.

20. A DNA construct containing a DNA sequence as claimed in claim 8 operably linked to regulatory regions capable of directing the expression or over-expression of a polypeptide having xylanase activity in a suitable expression host.

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

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 xylanase 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 xylanase activity in a suitable expression host.

24. A DNA construct conttining a DNA sequence as claimed in claim 17 operably linked to regulatory regions capable of directing the expression or over-expression of a polypeptide having xylanase activity in a suitable expression host, A transformed microbial host capable of the expression or over- expression of fungal xylanase harbouring the xylanase construct of claim

26. A transformed microbial host capable of the expression or over- expression of fungal xylanase harbouring the xylanase construct of claim 21. .27. A transformed microbial host capable of the expression or over- expression of fungal xylanase harbouring the xylanase construct of claim 22. o* 28. A transformed microbial host capable of the expression or over- expression of fungal xylanase harbouring the xylanase construct of claim 23.

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

30. A polypeptide having xylanase activity produced by expression using a 25 microbial host of claim

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

32. A polypeptide having xylanase activity produced by expression using a microbial host of claim 27. 33, A polypeptide having xylanase activity produced by expression using a microbial host of claim 28.

34. A polypeptide having xylanase activity produced by expression using a microbial host of claim 29. 29 Plusmid pNX-Tac lodged at the Australian Government Analytical Laboratories on March 17 1993 under accession number N93/12211. Dated this first day of July 1998 COMiMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION Patent Attorneys for the Applicant: F B RICE CO ee e *a*