YEAST STRAINS PRODUCING CELLULOLYTIC ENZYMES AND METHODS AND MEANS FOR CONSTRUCTING THEM

Abstract: Methods and means for the construction of strains of yeast capable of producing cellulolytic enzymes. This is achieved by the transfer of chromosomal genes or cDNA copies of mRNAs coding for cellulolytic enzymes isolated from the fungus Trichoderma reesei to yeast cells using recombinant DNA vectors capable of replicating in yeast. The correct expression of these cellulolytic genes in yeast leads to the production of cellulolytic enzymes which are secreted from the cell. This allows the yeast to hydrolyse beta -1,4-glucan substrates such as cellulose.

Description: YEAST STRAINS PRODUCING CELLULOLYTIC ENZYMES AND METHODS AND MEANS FOR CONSTRUCTING THEM BACKGROUND OF THE INVENTION Three different classes of enzymatic activity have been shown to be required for the complete hydrolysis of cellulose to glucose. The two major activities involved in cellulose solu- bilization are endoglucanase (EC 3.2.1.4) and cellobiohydrola (EC 3.2.1.91) (1, 2). For the production of glucose a third type of activity, cellobiase or g-glucosidase (EC 3.2.1.21) i also required. The precise manner in which these three differ classes of enzyme interact to bring about the complete hydro¬ lysis of cellulose is not yet clear. Same filamentous fungi produce a number of different isoenzym of each class of cellulolytic enzyme which apparently interac synergistically in hydrolysis (3, 4, 5, 6). • Trichoderma has been shown to produce at least two immunologi cally distinct cellobiohydrolases CBH I and CBH II, at least endoglucanases, ENDO II and ENDO III, and a β-glucosidase. Wh enzymatic hydrolysis of cellulose proceeds most rapidly in th presence of all these enzymes, CBH I alone is able to degrade crystalline cellulose to glucose and cellobiose (7, 8, 9). Two groups have reported the molecular cloning of the T. rees gene for CBH I and the complete sequence of this gene is know (10, 11). Yeast is an important industrial organism and is used for brewing, wine making, baking, ethanol production, single cell protein production and more recently for the production of pharmaceuticals such as interferon, growth hormone and Hepati B virus antigen. Yeast do not produce enzymes that degrade cellulose. The development of yeast strains able to hydrolyse cellulose would make possible improvements in existing process where cellulose or glucans are present in the raw material use As important would be the possibility of developing new proces not currently possible. In filtration and clarification of beer high molecular weight β-glucans originating from barley grain cause problems. In th brewing industry microbial β-glucanases are used to remove th β-glucans. If the yeast used in the production of beer were able to produce endoglucanases, the filterability of beer wou significantly be improved and the cost of filtering would decrease . By transferring individual fungal cellulase genes t yeast it is possible to produce yeast strains that produce on one cellulase enzyme. Such yeast strains would produce enzyme for use in, for example the pulp and paper industry. Cellulos used in paper making could be swelled by pretreating with one . cellulase enzyme, which would bring about swelling without excessive hydrolysis of cellulose. There -are two ways in which a foreign gene can be expressed i yeast. The simplest is to join the whole gene from the chromo of the donor organism to a yeast vector and transform a yeast cell. If the yeast genetic system recognizes the appropriate sequences in the transferred gene the gene will be expressed. However, in practice this is rare and depends at least in par the genetic distance between the donor organism and the yeast For example, of the five genes from Aspergillus niger tested in Saccharomyces cerevisiae, only one of these was found to express (12). Therefore it cannot be assumed that heterologou genes will automatically be expressed in yeast. The second method of obtaining expression of genes in yeast i by connecting either the chromosomal gene or a cDNA copy of t messenger RNA coding for the desired gene to a yeast promotor sequence. In this way, human eukaryote interferon (13), hepatitis B virus surface antigen (14), bovine rennin (15), and mouse α-amylase (16) have all been expressed in yeast. These and other studies show that while expression of the cDNA or gene is always obtained, the amount and cellular location o the product is very difficult to predict in the absence of experimentation. Montenecourt (1) outlined a number of possibl cloning strategies for cloning cellulase genes from T. reesei but did not describe the methods to be used to achieve the goa SUMMARY OF THE INVENTION In accordance with this invention described are yeast strains capable of producing cellulolytic enzymes, methods for construc tion of these strains, recombinant DNA vectors needed in the construction of these strains, methods used in the .constructio of these vectors, and cDNA copies of cellulolytic enzymes codin genes. Chromosomal genes coding for three different cellulases, CBH I, CBH II and ENDO II were isolated from a λ phage gene libary of T. reesei by differential hybridisation. Fragments of these genes were used to isolate full length cDNAs from a T. reesei cDNA libary. cDNAs for the three cellulases CBH I, CBH II and ENDO II and th CBH I gene were transferred to suitable 2 μ yeast plasmids. Whe used to transform suitable yeast strains, they directed the expression and secretin of the respective cellulase enzyme. The cellulases produced by the yeast were shown to have similar activities to the native fungal enzyme. A cellulolytic yeast strain Saccharomyces cerevisiae VTT-RC-840 produced in accordance with the present invention has been deposited in the National Collection of Yeast Cultures, Norwich United Kingdom, under the deposit number NCYC No. R 128 since April 6, 1984. BRIEF DESCRIPTION OF THE DRAWINGS The present invention is described below in greater detail wit reference to the accompanying drawings. Figure 1 shows the restriction map of T. reesei cellobiohydro- lase I (CBH I) chromosomal gene. The coding region is marked with thickened line. Figure 2 shows the restriction map of T. reesei cellobiohydro- lase II (CBH II) chromosomal gene. The coding region is marked with thickened line. Figure 3 shows the restriction map of T. reesei endoglucanase II (ENDO II) chromosomal gene. The coding region is marked wit thickened line. Figure 4 shows the construction of plasmid YEpNP03 for express of a chromosomal copy of CBH I gene from T. reesei in yeast. Figure 5 shows the cDNA sequence of the CBH II gene of T. rees from plasmid pTT09. •r Figure 6 shows the cDNA sequence of the ENDO II gene of T. ree from plasmid pTTll. The positions of introns found in chromoso mal copy of the gene are marked with arrow (+) . Figure 7 shows the construction of plasmid pMPll for expressio of T. reesei CBH I in yeast. Figure ά shows the construction of plasmid pMP29 for expressio of T. reesei CBH II in yeast. Figure 9 shows the construction of plasmid pMP311 for expressi of T. reesei ENDO II in yeast. Figure 10 shews the enzyme activity of CBH I produced by the yeast strain VTT-RC-84001. Detailed description The definitions used in this detailed description are as defin in the Giibert and Talmadge Patent (USA) 4,338,397. Materials Bacterial and fungal strains, plasmids, and phage. T. reesei strain VTT-D-80133, a mutant strain with improved production o cellulolytic enzymes derived from QM 9414 (17) after several successive mutation steps (18), was used for isolation of the genes from cellobiohydrolase I (CBH I), cellobiohydrolase II (CBH II) and endoglucanase II (ENDO II). Escherichia coli strains Q358 and Q359 and the phage λ 1059, used in the construction of the T. reesei gene bank were provided by Dr. J. Karn (19) . E. coli HB 101 was used as a hos in 5 transformation with the plasmid pBR 322. E. coli JM 101 and the phage M 13 mp 7 (20) and the plasmids pUC 8 and pUC 9 (24), used in the dideoxy sequencing, were from the laboratory F. Sanger . Yeast strains used were Saccharomyces cerevisiae 0L1 (Mata leu 2-3 leu 2-112 his 3-11 his 3-15 ura 3-251 ura 3-373) (22) and S. cerevisiae MT302-lc (Mata arg 5-6 leu 2-3 leu 2-112 " his 3-11 his 3-15 pep 4-3 ade 1) (23). A 12 kb cosmid p3030 obtained from Barbara Hohn, which replica both in E. coli and in yeast was used as vector for transferri the chromosomal copy of CBH I to yeast. Cosmid p3030 contains genes for ampicillin and tetracycline resistance in E. coli and the his3 gene for selection in yeast. The vector contains a cos site which enables it to be packaged into infective λ phage particles in vitro and the yeast 2 μ EcoD fragment. Yeast expression vector containing the phosphogl - * cerokinase (PGK) ge promoter was used for expression of the cDNA copies of cellula genes in yeast (23). Enzymes. Restriction enzymes were purchased from Amershain (UK), Boehringer Mannheim (FDR) and Bethesda Research Laboratories (Gaithersburg, MD) and used according to the manufacturers' instructions. T4 ligase and the DNA polymerase I large subunit were from Biolabs and the calf intestine phosphatase from Boehringer Mannheim. Reverse transcriptase was from Dr. J.W. Beard (Life Sciences Inc., St. Petersburg, Fla.). Protoplastin enzyme, Zymolyase 60000 was obtained from Kirin Brewery Co., Japan. Klenow fragment of E. coli polymerase I was from Boehringer Mannheim. General growth media. E. coli HB101 was grown in L-broth. Transformants were selected on L-plates supplemented with 1.5 agar and containing lϋϋ μg/ml ampicillin. The concentration of tetracycline added to L-plates was 10 μg/ml. Complete medium YPG for growth of yeast contained 1 % yeast extract, 2 % pepto and 2 % glucose. Yeast minimal medium, YMB, contained 0.67 % yeast nitrogen base (Difco, Detroit, USA) and 2 % sugar (lacto cellobiose, starch or glucose). The final concentration of ami acids added was as described (24). The solidifying agent on yeast plates was 2 % agar (Difco Bacto Agar). In yeast proto¬ plast plating medium 1.2 M sorbitol was added as an osmotic stabilizer. The top agar used in plating the yeast protoplasts for regeneration was prepared as minimal medium but using 3 % purified agar (Difco) as a solidifying agent. All methods unless otherwise specified are as described in Maniatis et al. 1982 (25). Isolation and characterization of the cellulolytic genes from the fungus T. reesei Polyadenylated (polyA + ) messenger RNA isolated from T. reesei rnycelia actively producing cellulases directs in the in vitro synthesis - in a rabbit reticulocyte lysate - of a number of large polypeptides that are precipitated by antibody prepared against purified cellulolytic enzymes. Messenger RNA isolated from repressed glucose grown mycelia does not direct the synthesis of these cellulase-specific polypeptides. This difference between induced and repressed populations was used to identify a collection of hybrid λ phages containing T. ree genes strongly expressed during production of cellulolytic enzymes. For the isolation of cellulase-specific, induced mRNAs T. ree (strain VTT-D-80133) was grown as described by Bailey and Nev lainen (26) except that the medium contained 2 % lactose and • 2 % of a soluble extract of distillers spent grain. Samples taken during cultivation were assayed for activity against dye Avicel, hydroxyethylcellulose (HEC) and for soluble protein (2 Estimation of reducing sugars was by the method of Surnner (27) Cellular RNA from mycelia was isolated by a modification of th method of Ohi and Short (28). The frozen mycelia was ground to a .fine powder under liquid nitrogen and suspended in a buffer containing 20 mM Tris-HCl (pH * 7.6). 0.1 M NH 1 M Mg (OAc) , 4 2 10 mM Na-iodoacetate, 0.5 mg/ml polyvinylsulfate and 2 % Na-dodecyl sulfate (SDS). Following incubation at 37 C for 30 minutes, insoluble material was removed by centrifugation a 13000 g for 10 minutes. The ρoly(A) + fraction was purified by chromatography through a oligo(dT) cellulose column (Bethesda Research Laboratories (29 and in vitro translation was carried out with a rabbit reticu¬ locyte lysate using 35 S-methionine (Amersha International Ltd (30). Immunoprecipitation was carried out according to Dobber- stein (31) using antiserum prepared against purified CBH I, CBH II or ENDO II, or with the corresponding preimmune serum. Table 1 shows the molecular weights of proteins precipitated b antiserum against specific cellulases analysed on 7.5-15 % SDS polyacrylamide gels (32). Table 1. Antiserum In vivo In vitro CBH I 71 000 67 000 CBH II 63 000 48 000 ENDO II 62 000 53 000 The construction of the T. reesei gene bank was carried out as follows. Conidia of Trichoderma reesei were. germinated in a liquid medium containing 1.5 % KH PO , 0.5 % (NH ) SO , 0.06 % 2 4 4 2 4 MgSO -7H 0, 0.06 % CaCl , 0.15 % proteose peptone, 0.03 % urea, 4 2 2 2 % sucrose and minimal salts. Cultures were incubated with o shaking at 29 C for about 12 h. The isolation of nuclei was carried out using a slightly modified method of Hautala et al. (33). DNA was isolated from a crude nuclear pellet obtained by differential centrifugation of homogenized mycelium. The crude nuclear pellet was treated with SDS-amylase solution (100 M EDTA pH 8.0, 140 mM NaCl, 1 % Nadecylsulfate and 3.3 % α-amyla o obtained from Merck, Darmstadt, FRG) for 1 h at 37 C. Proteina K (final concentration 0.8 % w/v) was then added and incubatio o was continued for 2 h at 37 C with gentle shaking. After incubation, cell debris was removed by centrifugation and DNA was precipitated from the supernatant with ethanol. The DNA wa then purified by CsCl centrifugation. The chromosomal DNA from T. reesei was partially digested with Mbol and sized by sucros density gradient centrifugation. Fifteen-20 kb fragments were ligated to Bam Hi-cleaved λ 1050 DNA. In vitro packaging of th recombinant molecules was carried out using packaging extracts prepared by the method of Hohn as described by Maniatis et al . (25). Recombinant phages were transferred from the agar to nitro¬ cellulose filters (Schleicher & Schϋll, BA 85) as described by Benton and Davis (34). cDNAs made from induced RNA (described earlier) and from mRNA isolated from fungus grown in the presen of glucose were used as probes. cDNA first strand synthesis was carried out by the procedure of Efstradiatis et al . (35) but using 10 uCi of 32 pαATP per 50 μl reaction. The in situ plaque hybridization was carried out according to Maniatis et al . (25) Hybridization was detected by autoradiography of the filters on Kodak X-OMAT film. Positive plaques were picked into 1 ml of 3M o (25) and a drop of chloroform and stored at -4 C. Hybrid phage hybridizing only to cDNA made with induced mRNA containing cellulase coding sequences were purified extensively and retested by hybridization to both probes . A number of different hybrid clones that hybridized strongly to the induced cellulase probe were identified and selected for further ana-lysis. The hybrid phages containing genes induced when the fungus produces cellulases were first grouped according to their restriction enzyme patterns. Then the particular cellulase gene in each group was identified by hybrid selection of messenger RNA. DBM paper was obtained from Schleicher and Schϋll (Keene, NH) and activated according to the maker's instructions. Binding of DNA to the activated paper and RNA hybridization and elution was carried out according to Maniatis et al. (25). RNA was translated with a rabbit reticulocyte lysate supplied by Amersh International Ltd. and the proteins produced were labeled with 35 S-methionine. The proteins were analysed by autoradiography on Kodak X-OMAT film after separation on a 7-15 % polyacrylamid gradient denaturing gel . The size of the proteins obtained from particular phage by hybrid selection and their cross reaction with specific antiser is shown in Table 2. Table 2. Single and double digests of the clone 44A, were analyzed on 0.6 % and 1.5 % agarose gels. The fragments were electrophoreti cally transferred to Gene Screen membranes (New England Nuclear MA) and hybridized to the induced cDNA probe as instructed by the manufacturer. This procedure permitted the construction of restriction enzyme maps of the three cellulose genes . These restrictions enzyme maps are shown in figures 1, 2 and 3. The nucleotide sequence of the CBH I, CBH II and ENDO II genes was generated by dideoxy sequencing (36) using restriction enzyme fragments or DNA fragments obtained by the "shotgun" procedure (37). The construction of a yeast vector containing the CBH I chromo somal gene The hybrid phage 44A (11) DNA containing the CBH I of Trichode reesei hypercellulolytic mutant strain VTT-D-80133, was digest with Pst I to give a mixture of fragments one of which being about 12 kb and containing the entire CBH I gene with its own regulatory sequences. The resulting DNA-fragments were ligated with the yeast cosmid p3030 digested partially with the same enzyme . The yeast strain OL1 was transformed to his + with the DNA- mixture described above. Transformation was carried out essen¬ tially as described by Gerbaud et al . (38). Transformed cells were plated on yeast minimal medium with leucin and uracil but lacking histidine.. The clones were further tested in situ plaque hybridization for the presence of the CBH I gene originated from T. reesei. The presence of an intact CBH I gene in yeast was ensured by isolating total DNA (39) from a transformant colony and digesti it''with restriction enzymes Bgl II and Hinc II. DNA was trans¬ ferred to nitrocellulose filter (40) from agarose gel and hybridized to a M13 probe (41) containing the 0.7 kb Eco Rl fra ment from CBH I gene. Figure 4 shows the construction of a hybr plasmid containing the CBH I gene . Isolation of full length cDNAs coding for the enzymes CBH I, ,CBH II and ENDO II A cDNA bank from T. reesei was made from induced mRNA isolated from cells as described earlier. However, after the frozen mycelia had been ground under liquid nitrogen it was suspended in 5 volumes a guanidinium isothiocyanate buffer as described by Maniatis et al . (25). The RNA preparation was then carried out as described (42). cDNA first strand synthesis was carried out according to Mania (25) and the second strand was carried out according to Gubler and Hoffmann (43). The double stranded cDNA was then treated w T -polymerase to give blunt ends and small cDNAs less than 500 nucleotides long removed by passage through a CL-4B column (Pharmacia) . Long cDNAs were then ligated to a Sma I digested and phosphatase treated preparation of pUC 8 vector. The ligat mixture was used to transform E. coli strain JM 105 and the cDNA bank was stored on nitrocellulose filters. Full length cDNAs coding for CBH I, CBH II and ENDO II were isolated from a cDNA bank using specific restriction fragments as probes. For the identification of CBH I, a radioactive Eco RI-Hind III fragment from the 5' end of the chromosomal gene w used to identify long cDNAs. A plasmid pTTOl from a clone containing sequences homologous to this Eco RI-Hind I ' ll fragme was further characterized by sequencing of the cDNA ends by double stranded dideoxy sequencing. 1 μg of purified plasmid w denatured in 0.4 M NaOH at room temperature for 5 minutes at a concentration of 100 ng/μl. 5 ul of sequencing or reverse sequencing primer (Amersham) was added and the mixture was pre cipitated with ethanol. After washing the pellet was resuspend in 10 μl at 14 mM Tris pH 8 - 7 mM MgCl . Sequencing reactions 2 were done according to general methods (36) except that temper o ture was kept at 37 C CBH II cDNAs were isolated using a Pvu II fragment from the 5' end of the chromosomal gene and the plasmid ρTT09 characterized as " for the CBH I cDNA. ENDO II cDN were identified using a Kpn I-Sal I fragment from the 5' end o the gene and plasmid pTTll also characterized as for the CBH I cDNA. All cDNAs were then sequenced to determine that their sequence corresponded to that of the gene from which they are transcribed. The DNA sequences of CBH II and ENDO II cDNAs are shown in Figures 5 and 6. The cDNA sequence of CBH I was identical to that already described (10). The construction of expression vectors containing cDNAs for th production of fungal cellulases in yeast The efficient yeast expression vector pMA 91 has been assemble using the regulatory sequences of the yeast phosphoglycerokina (PGK) gene (23). The sequences coding for the amino acid seque of the enzyme have been removed from the gene and replaced by single Bgl II site. This deleted gene has then been inserted i a yeast/coli shuttle plasmid. a) CBH I expression vector (Figure 7) The CBH I cDNA was removed from plasmid (pTTOl Figure 7) by digestion with Hinc II and the cDNA fragment isolated from an agarose gel. pMA 91, the expression vector was cleaved with Bgl II and the ends were filled in with the Klenow fragment. The vector was treated with phosphatase, ligated to the cDNA and transformed into E. coli strain HB101 by selection for expression of the vector leucine gene (Figure 7). Plasmid DNA was isolated from num fber of transformants and those clones containing o the cDNA insert in the correct orientation with respect to the PGK pro- motor - as identified by restriction enzyme analysis - were retained. DNA from one of these clones (pMP 11) was then trans¬ formed into yeast strain MT 302-lc by the method described earlier by selection of the le ' ucine marker of pMA 91 resulting in strain VTT-RC-84011. b) CBH II expression vector (Figure 8) CBH II cDNA was removed from plasmid pTT 09 using Eco Rl and Bam HI. The ends of the DNA were filled in with Klenow fragment The cDNA fragment was then isolated from an agarose gel and ligated to the vector pMA 91 prepared as for CBH I. The ligation mix was transferred into HB101 and clones containi the cDNA in the correct orientation identified. Figure 8 shows the DNA sequence at the junctions between pMA 91 and the cDNA. Plasmid pMP 29 with the cDNA in the correct orientation was th used to transform yeast MT302-lc by selection for the leucine marker to give strain VTT-RC-84012. c) ENDO II expression vector (Figure 9) The ENDO II cDNA was tranferred to pMA 91 exactly in the same way as CBH II cDNA. Figure 9 shows the DNA sequences at the junctions between pMA 91 and the ENDO II cDNA. The plasmid, pMP 311 containing the * ENDO II cDNA in the correct orientation was transferred to yeast as described earlier to give strain VTT-RC-84013. Culturing the hybrid yeast strains to produce the cellulolytic enzymes, CBH I, CBH II and ENDO II Strain- VTT-RC-84001 containing YEpNP03 was grown in a yeast minimal medium with leucine and uracil for three days after wh complete medium (1/3 volume) was then added to allow the cells pass through one more division. Strains VTT-RC-84011 (CBH I cDNA) , VTT-RC-84012 (CBH II cDNA) VTT-RC-84013 (ENDO II cDNA) were grown in a yeast minimal medi containing arginine, histidine and adenine for three days afte which complete medium 1/3 volume was added to allow the ceells pass through one more division. The final volume of the cultur was about 150 ml. Preparation of different fractions for analysis of the locatio of enzyme activity Three fractions were prepared from hybrid yeast cultures for analysis of enzyme activity. Fraction 1 comprised the growth medium without the cells. Fraction 2 comprises the supernatant left when protoplasts are pelleted and fraction 3 comprises th supernatant of lysed protoplasts. After cultivation yeast cells were collected by centrifugation and the supernatant was saved (Fraction 1). The resulting pell was washed twice with distilled water and 1.2 M sorbitol . The pellet was then resuspended in protoplasting buffer (1.2 M sor bitol, 10 mM Tris and 10 M CaCl, pH 7.6) and Zymolyase 60000 added at a concentration of 30 μg/ml of protoplasting suspensio o Suspension was incubated in a waterbath at 37 C for 60 minutes with gentle shaking. The protoplasts so formed were pelleted a the resulting supernatant (periplasmic cell contents) (Fractio 2) saved for enzyme activity determinations. In some cases fractions 1 and 2 were concentrated by ultrafiltration (Amicon) Protoplast pellets were washed with cold 1.2 M sorbitol and resuspended in 1.2 ml of 5 mM citrate buffer pH 5.0, pelleted and the supernatant was saved (Fraction 3). " Measurement of cellulase enzyme activity produced by the hybrid yeasts 1 CBH I activity from VTT-RC-84001 The three different fractions were tested for CBH I enzyme activity using amorphous ball milled cellulose which is attache only by cellobiohydrolases (44). The total protein concentratio of the samples was about 300 μg/ml. Hydrolysis of the substrate caused by active cellobiohydrolase enzyme was measured by following the change in absorbance at 620 nm. CBH I type activi was found only in fraction 2, the periplasmic or intramural space . Figure 10 shows the activity of the CBH I enzyme produced by th yeast strain VTT-RC-84001 and secreted into the intramural spac as compared to the control yeast containing only vector p3030 DNA and 1 μg of Trichoderma CBH I. This Figure shows that the hybrid yeast strain produces active CBH I which appears to be o at least as resistant to incubation at 50 C for 3 days as in the native enzyme. The CBH I produced by the yeast represents 1-2 % of the protein of intramural space protein. As the intron sequences of fungus are different from those of yeast it is not likely that yeast would process off the fungus gene introns. Probably for that reason the product of the chromosomal gene remains in the yeast periplasmic space and is not secreted from the cell as is the product coded by the cDNA sequence. This result suggests that transferring the chromosom gene coding for CBH I to yeast, results in the production of a smaller protein, which, however, has the same type of activity as the full length cellulase. 2 CBH I activity from VTT-RC-S4011 The three different fractions were tested for CBH I enzyme activity as ' just described. Hpwever, in this case, most of the CBH I type activity was found in the growth medium. The result with a final protein concentration during hydrolysis of 5 μg/m is very similar to that shown in Figure 10. The CBH I enzyme produced with this construction represented 1-5 % of total cel •r protein. 3 CBH II activity from VTT-RC-84012 The three different fractions .were tested from cellobiohydrola activity as described for strain VTT-RC-84001. As with strain VTT-RC-84011, most of the cellobiohydrolase type activity was found in the growth medium. The results with a final protein concentration of 10 μg/ml is similar to that shown in Figure 1 The CBH II enzyme produced with this construction represented 1-5 % of total cell protein. 4 ENDO II activity from VTT-RC-84013 The three different fractions were tested for endoglucanase o activity by following the hydrolysis of 0.1 % β-glucan at 50 C The reducing sugars liberated in 5 minutes (overnight) were measured as glucose using the dinitro salisylic acid method (4 Most, of the ENDO II activity was found secreted into the growt medium. The ENDO II enzyme produced with this construction represented 1-5 % of total cell protein. It is considered that the invention and many of its attendant advantages will be understood from the foregoing description and that it will be apparent that various changes may be made in the steps of the described method for mature protein synthesis v/ithout departing from the spirit and scope of the invention or sacrificing all of its material advantages, the method herein before described being merely a preferred embodi ment. • REFERENCES 1. B.S. Montenecourt, 1983, Technol. in Biotechnology l_ t 156-161. 2. ΞNARI, T-M. Microbiol . Cellulases, p. 183-223. In: Microbiol. Enzymes and Biotechnologes. William M. Fog (ed.). Applied Science Publishers, London and New Yor 3. Shoemaker, S.P. and Brown, R.D. Jr. (1978a) Biochim. Biophys. Acta 523, 133-146. 4. Shoemaker, S.P. and Brown, R.D. Jr. (1978b) Biochim. Biophys. Acta 523, 147-161. 5. Fagerstam, L. and Petterson, L.G. (1979) FEBS Lett. 363-367. 6. Fagerstam, L. and Petterson, L. G. (1980) FEBS Lett. 97-100. 77 " Sprey, B. and Lambert, C. (1983) FEMS Microbiol. Lett 18, 217-222. 8. Chanzy, H., Henrissat, B., Vuong, R. and Schulein, M. (1983) FEBS Lett..153, 113-118. 9. Nummi, M. , Niku-Paavola, M.L., Lappalainen, A., Enari, T.M. and Raunio, V. (1983) Biochem. J. 215, 677-683. 10. Shoemaker, S., Schweickart, V., Ladner, M. , Gelfand, Kwok, S., Myarαbo, K. and Innis, M. (1983) Biotechnol. 691-696. 11. Teeri, T., Salovuori, I. and Knowles J. (1983), Bio- technol. 1, 696-699. 12. Penttila, M.E., Nevalainen, K.M.H., Raynal, A. and Knowles, J.C.K. 1984: Molec. , Gen. Genetics, in press 13. Hitzeman, R.A. , F.E. Hagie, H.L. Levine, D.V. Goeddel Ammerer & B.D..Hall: Expression of a human gene for interferon in yeast. Nature 293, 717-722 (1981). 14. Valenzuela, P.,A. Medina, W.J. Rutter, G. Ammerer & B.D. Hall: Synthesis and assembly of hepatitis B viru surface antigen particles in yeast. Nature 298, 347-3 (1982) . 15. Mellor, J., M.J. Dobson, M.A. Roberts, M.F. Tuite, J.S. Emtage, S. White,- P.A. Lowe, T. Patel, A.J. Kingsman & S.M. Kingsman: Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae. Gene 24 (1983): 1-14. 16. K.K. Thomsen, 1983, Carlsberg Res. Commun. 48_ p. 545-555. 17. Mandels, M. , Weber, J. and Parrek, R. (1971) Appl . Microbiol. 2l_, 152-154. 18. Nevalainen K.M.H. 1981: Appl. Environ. Microbiol. 41: 595-596. 19. Karn, J., Brenner, S., Barnett, L. , and Cesaveni, G. 1980. Novel bacteriophage λ cloning vector. Proc. Natl Acad. Sci. 77:5172-5176. 20. Messing, J., Crea, R. , and Seeburg, P.H. 1981. A syste for shotgun DNA sequencing. Nucleic Acids Res. 9: 309-321. 21. Vieira, J. and Messing, J. 1982: Gene 19:259-268. 22. Boy-Marcotte, E., Jaquet, M, (1982) A dictyostelium discoideum DNA fragment complements a Saccharomyces cerevisiae ura3 mutant. Gene 20:433-440. 23. Mellor, J., Dobson, M.J., Roberts, N.A. , Tuite, M.F., Emtage, J.S., White, S., Lowe, P.A., Patel, T., Kingsman, A.J. and Kingsman, S.M. 1983. Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae. Gene 24: 1-14. 24. Sherman, F., Fink, G.R. and Hicks, J.B. 1981. Methods in yeast genetics. Cold Spring Harbor Laboratory, New York. p. 62. 25. Maniatis, T. , Fritsch, E.F. and Sambrook, J. 1982. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 26. Bailey, M.J. and Nevalainen, K.M.H. 1981. Induction, isolation and testing of stable Trichoderma reesei * mutants with improved production of solubilizing cellulase. Enzyme Microb. Techno1. 3:153-157. 27. Sumner, J.B. and Somers, G.F. (1949) in Laboratory experiments in biological chemistry 2nd ed. pp. 38-39, Academic Press, New York. 28. Ohi, S., and Short, J. 1980. A general procedure for preparing messenger RNA from eukaryotic cells without using phenol. J. Appl. Microbiol. 2:398-413. 29. Aviv, H. and Leder, P. (1972) „Proc. Natl. Acad. Sci. 1408-1412. 30. Pelham, H.R.3. and Jackson, R.J. (1976) Env. J. Eioch 67 pp. 247-266. 31. Dobberstein, B. , Garoff, h. and Wawen, G. (1979) Cell 17_, 759-769. 32. Laemmli, U. 1970. Cleavage of structural proteins dur the assembly of bacteriophage T4. Nature 227: 680-685 33. Hautala, J.A., Corner, B.H., Jacobson, J.W., Patel, G and Giles, N.H. 1977. Isolation and characterization nuclei from Neurospova crassa. J. Bacterial. 130: 704-713. 34. Benton, W.D. and Davis, R.W. 1977. Screening λ gt recombinant clones by hybridization to single plaques in situ. Science 196:180-182. 35. Efstradiatis, A., Kafatos, F.C., Maxam, A.M. and Maniatis, T. 1976. Enzymatic in vitro synthesis of gl genes. Cell 7:279-288. 3.6. Sanger, F., Nicklen, S. and Coulson, A.R. 1977. Proc. Natl. Acad. Sci. USA 74:5463-5467. 37. Deiniger, P.L. 1983: Anal. Biochem. 129: 21b-223. 38. Gerbaud, C. , Faurnier, P., Blanc, H. , Aigle, M. , Heslot, H. and Guerinau, M. 1979. Gene 5_:233-253. 39. Sherman F. Fink, G.R. and Hicks, J.B. 1981: Methods i yeast genetics. Cold Spring Harbor Laboratory, New Yo pp. 77-80. 40. Southern, E.M. 1975. J. Mol. Biol . 98_: 503-517. 41. Hu, N. and Messing, S. 1982. Gene 17:271-277. 42. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, V/.J. 197 ► Isolation of biologically active ribonucleic acid from sources enriched m ribonucleas Biochemistry lb: 5294-5299. 43. Gubler and Hoffman 1983: Gene 25:263 44. Nun-mi, M. , Fox, P.C., Niku-Paavola, M.L. , and Enari, 1981 Anal. Biochem. 116:133-136. 45. Kirsop, B.H. 1953: J. Inst. Brewing 59:378

Claims: WHAT IS CLAIMED IS: 1. A DNA sequence which codes for a fungal cellulase enzyme, or its single or multiple base substitutions, deletions, inser tions, or inversions, which is derived from natural, synthetic or semi-synthetic sources and which is capable, when correctly combined with an expression vector, of expressing a non-native protein having cellulolytic activity upon transformation of a host organism by the vector, said DNA sequence coding for substantially the following amino acid sequence: HetneValGlyneLeuThrThrLeuAlaThrLeuAlaThr euAlaAlaSerValProLeuGluGluArgGlπAlaCysSerSerVal TrpGlyGlnCysGlyGlyGlπAsπTrpSerGlyProThrCysCysAlaSerGlySerThrCysValTyrSerAsnAspTyrTyrSerGIn CysLeuPpoGlyAlaAlaSerSerSepSerSerThrArgAlaAVaSerThrTI-rSerArgValSerProThrThrSerApgSerSerSer AlaThrProProPpoGlySerThpThrThrArgValProProValG1ySerG1yThrA1aThrTyrSerGlyAsnProPheValG1yVal ThrProTrpAl aAsnAl aTy pTyrAl aSerGl uVal SerSerLeuA" al 1 eProSerLeuThrG ' yAl aMetA " aThrAl aAl aAl aAl a ValAlaLysValProSerPheHetTrpLeuAspT rLeuAspLysT rProieuMetGluGlnThrLeuAlaAspneArgThrAlaAsn LysAsnGlyGlyAsnTyrAlaGlyGlnP eValVaUyrAspLeuProAspArgAspCysAlaAlaLeuAlaSerAsnGlyGluTyrSer neAlaAspGlyGlyValAlaLysTyrLysAsnTyrneAspT pIleApgGlnneValValGluTyrSerAspneArgThrLeuLeu Val'leGluProAspSerLeuAlaAsπleuValThrAsnL-uGlyThrProLysCysAl aAsnAl aGlπSerA ' aTyrLeuGluCysI le • AsnT PAI aVal Th p Gl nLeuAsnLeuP p oAsnV^l AT aMetTypLeuAspAl aGlyHi sAl aGlyTppLeuGlyTppPpoAl aAsπGl π AspPPOAlaAlaGlnLeuPheAlaAsnValTyrtysAsnAlaSe p Se p PPoA p gAlaLeuA p gGlyUeuAlaThrAsnValAlaAsnTyp AsnGlyT p pAsnneTh p Se p P P OP p oSe p Ty p Th p GlnGlyAsnAlaValTy p AsnGluLysLeuTy p IleHisAlalleGlyP p oLeu LeuAlaAsnHisGlyT p pSe p AsnAlaPheP elleTh p AspGlnGlyApgSe p GlyLysGlnPPoThrGlyGlnGlnGlnT p pGlyAsp TppCysAsπVallleGlyTh p GlyP eGlyneA p gP p oSerAlaAsnT p GlyAspSe p Leu euAspSe p PheValT p pValLysPPO GlyGlyGluCysAspGlyThpSe p AspSerSepAlaP p oArgPheAspSerHfsCysAlaLeuProAspΛlaLeuGlnPPoAlaPpoGlπ AlaGlyAlaT p pP eGlπAlaTy p PheValGlnLeuLeuTh p AsnAlaAsnPpoSepPheLeu or a portion thereof. 2. A DNA sequence according to Claim 1 which codes for the cellobiohydrolase II activity and has substantially the follow¬ ing nucleotide sequence : CTTG ' TAAGATCACCCTCTGTGTATTGCACC ATGATTGTCGGCATTCTCACCACGCTGGCTACGCTGGCCACACTCGCAGCTAGTGTGCCTCTAGAGGAGCGGC'AAGCTTGCTCAAGCGTC TGGGGCCAATGTGGTGGCCAGAATTGGTCGGGTCCGACTTGCTGTGCTTCCGGAAGCACATGC ' GTCTACTCCAACGACTATTACTCCCAG TGTCTTCCCGGCGCTGCAAGCTCAAGCTCGTCCACGCGCGCCGCGTCGACGACTTCTCGAGTATCCCCCACAACATCCCGGTCGAGCTCC GCGACGCCTCCACCTGGTTCTACTACTACCAGAGTACCTCCAGTCGGATCGGGAACCGCTACGTATTCAGGCAACCCTTTTGTTGGGGTC ACTCCTTGGGCCAATGCATATTACGCCTCTGAAGTTAGCAGCCTCGCTATTCCTAGCTTGACTGGAGCCATGGCCACTGCTGCAGCAGCT TCGCAAAGGTTCCCTCTTTTATGTGGCTAGATACTCTTGACAAGACCCCTCTCATGGAGCAAACCTTGGCCGACATCCGCACCGCCAAC AAGAATGGCGGTAACTATGCCGGACAGTTTGTGGTGTATGACTTGCCGGATCGCGATTGCGCTGCCCTTGCCTCGAATGGCGAATACTCT ATTGCCGATGGTGGCGTCGCCAAATATAAGAACTATATCGACACCATTCGTCAAATTGTCGTGGAATAnCCGATATCCGGACCCTCCTG GTTATTGAGCCTGACTCTGTTGCCAACCTGGTGACCAACCTCGGTACTCCAAAGTGTGCCAATGCTCAGTCAGCCTAC TTGAGTGCATC AACTACGCCGTCACACAGCTGAACCTTCCAAATGTTGCGATGTATTTGGACGCTGGCCATGCAGGATGGCTTGGCTGGCCGGCAAACCAA GACCCGGCCGCTCAGCTATNGCAAATGTTTACAAGAATGCATCGTCTCCGAGAGCTCTTCGCGGATTGGCAACCAATGTCGCCAACTAC AACGGGTGGAACATTACCAGCCCCCCATCGTACACGCAAGGCAACGCTGTCTACAACGAGAAGCTGTACATCCACGCTANGGACCTCTT CTTGCCAATCACGGCTGGTCCAACGCCTTCTTCATCACTGATCAAGGT ' CGATCΞGGAAAGCAGCCTACCGGACAGCAACAGTGGGGAGAC TGGTGCAATGTGATCGGCACCGGA * RGGTATTCGCCCATCCGCAAACACTGGGGACTCGTTGCTGGATTCG * RGTCTGGGTCAAGCCA GGCGGCGAGTGTGACGGCACCAGCGACAGCAGTGCGCCACGATTTGACTCCCACTGTGCGCTCCCAGATGCCTTGCAACCGGCGCCTCAA GCTGGTGCTTGGΠCCAAGCCTACTΠGTGCAGCTTCTCACAAACGCAAACCCATCGNCCTGTWGGCTTTCGTGACCGGGCTTCAAAC AATGATGTGCGATGGTGTGGTTCCCGGTTCGCGGAGTCΠTGTCTACTTTGGTTGTCTGTCGCAGGTCGGTAGACCGCAAATGAGCAACT GATGGATTGTTGCCAGCGATACTATAATTCACATGGATGGTCTTTGTCGATCAGTAGGCTAGAGAGAGAGAGAGAACATCTATCCACAAT GTGβAGTGTCTATT or a portion thereof. 3. A DNA sequence according to Claim 1 wherein the DNA is cDNA copy of mRNA coding for cellobiohydrolase II in Tricho¬ derma reesei. 4. A signal sequence responsible of the secretion of a proteinaceous material extracellularly, said signal sequence having substantially the following amino acid sequence: Met!!eValGlylleLeuT p Th p euAIaThrLeuAlaThpLeuAlaAlaSepYa * Ppo uGluG * uApg 5. A DNA sequence which codes for a fungal cellulase enzyme, or its single or multiple base substitutions, deletions, inser tions, or nversions, which is derived from natural, synthetic or semi-synthetic sources and which is capable, when correctly combined with an expression vector, of expressing a non-native protein having cellulolytic activity upon transformation of a host organism by the vector, said DNA sequence coding for substantially the following amino acid sequence:. MetAl aPpoSepValThPleuPpoLeuThpT PA" all eLeuAl all eA " aApgLeuVal Al aAl aGl nGI nPpoGlyThPSe p Th p PPO GluValHisPpoUysLeuThrThpTypLysCysThPLysSepGlyGlyCysValAlaGlnASpT rSepValValLeuAspTppAsnTyp A p gT p pMetHisAspAlaAsnTyrAsnSepCysT"pValAsπG1yG1yVa sπThpThpLeuCysPPθAspGluA1aThpCysG1yLys AsnCysPI-eπeGlnGlyValAspTy p AlaAlaSe p GlyValTh p TI- p SepGlySepSβPteuT rMetAsπGlnTyrMetPpoSepSβr SerG1yG " yTy p Se p Se p Va1Se p pPθA p gLeuTyrLeu euAspSerAspGlyG1uTy p ValMetLeuLysLeuAsπG1yGlπG1u eu SepPheAspValAspLeuSepA1a euPPθCysG1yGluAsnG1ySepLeuTyrLeuSePGlnMe AspGluAsnG1yGlyAlaAsnG " π TyrAsπT P AlaGlyAlaAsnTyrG1ySe p GlyTy p CysAspAlaG1nCysPPθVa1G1πThpTrpApgAsπGlyT pLeuAsπThrSep HisGlπGlyGlπGlyP eCysCysAsnGluMetAspIleLeuGluGlyAsnSerArgAlaAsπAlaLeuThPPPOHisSerCysTI-rAla ThrAlaCysAspSepAlaGlyCysGlyP eAsnProTy p GlySepGlyTypLysSepTypTypGlyPPoGlyAspThrValAspT p Ser LysTh p PheThPlleneTh p GlnP eAsnTh p AspAsnGlySepProSepGlyAsnLeuValSepneThrApgLysTypGln Yal Aspll ePPoSepAl aGl nPpoGlyGlyAspThrll eSepSerCy sPPoSepAl aSerAl aTy rGlyGlyLeuA " aThrMetGly y s AlaLeuSerSepGlyMetValLeuValPheSerlleT p pAsnAspAsnSerGlπTyrMe AsπT p LeuAspSerGlyAsπAlaGlyPpo CysSepSe p ThrGluGlyAsnPPoSe p AsnneLeuAlaAsnAsnPpoAsnThpHisValValPheSe p AsnlleA p gTppGlyAspne Gl y€ePThpThpAsnSepThp SepSepT pT pSepSβPSe p PPoSerCy sTh p Gl nTh p Hi sT p pGlyGI nCys GlyGlylleGlyTypSβpGlyCysLysThrCysThpSerGlyT rThr ysGlnTypSepAsnA-pTypTy p Se p GlnCysLeuxxx or a portion thereof. 6. A DNA sequence according to Claim 5, which codes for the endoglucanase II activity and has substantially the following nucleotide sequence: CCCCCCTATCTTAGTCCTTCTTGTTGTCCCAAA ATGGCGCCCTCAGTTACACTGCCGTTGACCACGGCCATCCTGGCNATTGCCCGGCTCGTCGCCGCCCAGCAACCG ' GGTACtAGCACCCCC GAGGTCCATCCCAAGTTGACAACCTACAAGTGTACAAAGTCCGGGGGGTGCGTGGCCCAGGACACCTCGGTGGTCCTTGACTGGAACTAC CGCTGGATGCACGACGCAAACTACAACTCGTGCACCGTCACCGGCGGCGTCAACACCACGCTCTGCCCTGACGAGGCGACCTGTGGCAAG AACTGCTTCATCCAGGGCGTCGACTACGCCGCCTCGGGCGTCACGACCTCGGGCAGCAGCCTCACCATGAACCAGTACATGCCCAGCAGC TCTGGCGGCTACAGCAGCGTCTCTCCTCGGCTGTATCTCCTGGACTCTGACGGTGAGTACGTGATGCTGAAGCTCAACGGCCAGGAGCTG AGCTTCGAI.GTCGAC'CTCTCTGCTCTGCCGTGTGGAGAGAACGGCTCGCTCTACCTGTCTCAGATGGACGAGAACGGGGGCGCCAACCAG TATAACACGGCCGGTGCCAACTACGGGAGCGGCTACTGCGATGCTCAGTGCCCCGTCCAGACATGGAGGAACGGCACCCTCAACACTAGC CACCAGGGCCAGGGCTTCTGCTGCAACGAGATGGATATCCTGGAGGGCAACTCGAGGGCGAATGCCTTGACCCCTCACTCTTGCACGGCC ACGGCCTGCGACTCTGCCGGTTGCGGCTTCAACCCCTATGGCAGCGGCTACAAAAGCTACTACGGCCCCGGAGATACCGTTGACACCTCC AACACCΤΤCACCATCATCACCCAGTTCAACACGGACAACGGCTCGCCCTCGGGCAACCTTGTGAGCATCACCCGCAAGTACCAGXXXXXX GTCGACATCCCCAGCGCCCAGCCCGGCGGCGACACCATCTCGTCCTGCCCGTCCGCCTCAGCCTACGGCGGCCTCGCCACCATGGGCAAG GCCCTGAGCAGCGGCATGGTGCTCGTGT CAGCATTTGGAACGACAACAGCCAGTACATGAACTGGCTCGACAGCGGCAACGCCGGCCCC TGCAGCAGCACCGAGGGCAACCCATCCAACATCCTGGCCAACAACCCCAACACGCACGTCGTCTTCTCCAACATCCGCTGGGGAGACATT GGGTCTACTACGAACTCGACT X X.. ' GAGCTUGACGACTTCGAGCAGCCCGAGCTGCACGCAGACTCACTGGGGGCAGTGC GGTGGCATTGGGTACAGCG " GGTGCAAGACGTGCACGTCGGGCACTACGTGCCAGTATAGCAACGACTACTACTCGCAATGCCTTTAGAGC GTTGACTTGCCTCTGGTCTGTCCAGACGGGGGCACGATAGAATGCGGGCACGCAGGGAGCTCGTAGACATTGGGCTTAATATATAAGACA TGCTATGTTGTATCTACATTAGCAAATGACAAACAAATGAAAAAGAACTTATCAAGC or a portion thereof. 7. A DNA sequence according to Claim 5, wherein the DNA is cDNA copy of mRNA coding for endoglucanase II in Trichoderma reesei. 8. A signal sequence responsible of the secretion of a proteinaceous material extracellularly, said signal sequence having substantially the following amino acid sequence: Me AlaP p oSe p VaUhPLeuProLeuThrTh p A * alleLeuAlalleA * aA p gLeuValAlaAlaGln 9. A recombinant DNA vector comprising a DNA sequence accord ing to any of Claims 1-3 or any of Claims 5-7 or a signal sequence according to Claim 4 or 8, said vector being able to replicate and express in a suitable host organism. 10. A recombinant DNA vector according to Claim 9 wherein the DNA sequence of Claims 1—3 is ligated to plasmid pMA 91. 11. A recombinant DNA vector according to Claim 10 said vecto being pMP 29. 12. A recombinant DNA vector according to Claim 9 wherein the DNA sequence of Claims 5-7 is ligated to plasmid pMA 91. 13. A recombinant DNA vector according to Claim 12 said vecto being pMP 311. 14. A recombinant DNA vector according to any. of Claims 9-13, which is able to replicate and express in yeast. 15. A recombinant DNA vector according to any of Claims 9-14, which is able to replicate and express in genus Saccharomyces. 16-< A recombinant DNA vector comprising the following combina tion: chromosomal gene isolated from Trichoderma reesei coding for cellobiohydrolase I ligated to plasmid p3030, or cDNA copy of mRNA isolated from T. reesei coding for cellobiohydrolase I ligated to plasmid pMA 91, said vector being able to replicate and express in yeast. 17. A recombinant DNA vector according to Claim 16, wherein the chromosomal gene coding for cellobiohydrolase I is ligated to the restriction enzyme cleavage site of partially digested Pst I of plasmid p3030 and wherein the vector is YEpNP03. 18. A recombinant DNA vector according to Claim 16, wherein the cDNA sequence coding for cellobiohydrolase I is ligated to the restriction enzyme cleavage site of Bgl II of plasmid pMA and is under the control of the regulatory regions of phospho- glycerokinase gene, and wherein the vector is pMP 11. 19. A recombinant DNA vector according to Claim 16, said vect being able to replicate and express in genus Saccharomyces. 20. A yeast strain containing one or several of the following DNA sequences: a DNA sequence according to any of Claims 1-3 or a DNA sequenc according to any of Claims 5-7 or a signal sequence according Claim 4 or 8 or a chromosomal gene or cDNA sequence coding for cellobiohydrolase I. 21. A yeast strain according to Claim 20 , which comprises a recombinant DNA vector containing the DNA sequence according t any of Claims 1-3. 22. A yeast strain according to Claim 21, wherein the DNA sequence of Claims 1-3 is ligated to plasmid pMA 91 and said reeombinant DNA vector being pMP 29. 23. A yeast strain according to Claim 20, which comprises a recombinant. DNA vector containing the DNA sequence according t any of Claims 5-7. 24. A yeast strain according to Claim 23, wherein the DNA sequence of Claims 5-7 is ligated to plasmid pMA 91 and said recombinant DNA vector being pMP 311. 25. A yeast strain according to Claim 20, which contains the recombinant DNA vector YEpNP03, wherein the chromosomal gene isolated from T. reesei and coding for cellobiohydrolase I is ligated to plasmid p3030. 26. A yeast strain according to Claim 20, which contains the recombinant DNA vector pMP 11, wherein the cDNA sequence isola from T. reesei and coding for cellobiohydrolase I is ligated to plasmid pMA 91. 27. A yeast strain according to any of Claims 20-26, said yea strain being a member of genus Saccharomyces. 28. A yeast strain according to any of Claims 20-27, said yea strain comprising: S. cerevisiae VTT-RC-84001 (NCYC R 128) S. cerevisiae VTT-RC-84011 S. cerevisiae VTT-RC-84012 S. cerevisiae VTT-RC-84013. 29. A method for constructing a recombinant DNA vector accord to Claim 9 or 16, said method comprising ligating a DNA sequen according to any of Claims 1-3 or 5-7 or a signal sequence according to Claim 4 or 8 to a plasmid able to replicate and express in yeast or ligating a chromosomal gene coding for cellobiohydrolase I to plasmid p3030 or a cDNA sequence coding for cellobiohydrolase I to plasmid pMA 91. 30. A method for constructing a yeast strain according to Claim 20, said method comprising transforming a yeast strain by one or several of the recombinant DNA vectors of Claims 9-19

Inventors: KNOWLES JONATHAN, LEHTOVAARA-HELENIUS PAEIVI, NEVALAINEN HELENA, PENTTILAE MERJA, SALOVUORI IRMA, TEERI TUULA, KNOWLES, JONATHAN, LEHTOVAARA-HELENIUS, PAEIVI, NEVALAINEN, HELENA, PENTTILAE, MERJA, SALOVUORI, IRMA, TEERI, TUULA
Applicants: VALTION TEKNILLINEN, VALTION TEKNILLINEN TUTKIMUSKESKUS
Application year: 1985
Main classification: C12N15
IPC codes: C12N9, C12N15