| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 09.01.2001 |
| PATENT TITLE |
Heterologous polypeptides expressed in filamentous fungi, process for making same, and vectors for making same |
| PATENT ABSTRACT |
Novel vectors are disclosed for expressing and secreting heterologous polypeptides from filamentous fungi. Such vectors are used in novel processes to express and secrete such heterologous polypeptides. The vectors used for transforming a filamentous fungus to express and secrete a heterologous polypeptide include a DNA sequence encoding a heterologous polypeptide and a DNA sequence encoding a signal sequence which is functional in a secretory system in a given filamentous fungus and which is operably linked to the sequence encoding the heterologous polypeptide. Such signal sequences may be the signal sequence normally associated with the heterologous polypeptides or may be derived from other sources. The vector may also contain DNA sequences encoding a promoter sequence which is functionally recognized by the filamentous fungus and which is operably linked to the DNA sequence encoding the signal sequence. Preferably functional polyadenylation sequences are operably linked to the 3' terminus of the DNA sequence encoding the heterologous polypeptides. Each of the above described vectors are used in novel processes to transform a filamentous fungus wherein the DNA sequences encoding the signal sequence and heterologous polypeptide are expressed. The thus synthesized polypeptide is thereafter secreted from the filamentous fungus. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | November 24, 1997 |
| PATENT REFERENCES CITED |
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Genet., 199:37-45 (1985). McKnight, G. L., et al., "Nucleotide Sequence of the Triosephosphate Isomerase Gene from Aspergillus nidulans:Implications for a Differential Loss of Introns", Cell, 46:143-147 (1986). Ballance, D. J., et al., "Gene cloning in Aspergillus nidulans:isolation of the isocitrate lyase gene (acuD)", Mol. Gen. Genet., 202:271-275 (1986). Orbach, M. J., et al., "Cloning and Characterization of the Gene for .beta.-Tubulin from a Benomyl-Resistant Mutant of Neuropora crassa and Its Use as a Dominant Selectable Marker", Molecular and Cellular Biology, 6:2452-2461 (1986). Caddick, M. X., et al., "Cloning of the regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans", The EMBO Journal, 5(5):1087-1090 (1986). Vollmer, S. J., et al., "Efficient cloning of genes of Neurospora crassa", Proc. Natl. Acad. Sci. USA, 83:4869-4873 (1986). Newbury, S. F., et al., "Sequence analysis of the pyr-4 (orotidine 5'-P decarboxylase) gene of Neurospora crassa", Gene, 43:51-58 (1986). Huiet, L., et al., "The qa repressor gene of Neurospora crassa:Wild-type and mutant nucleotide sequences", Proc. Natl. Acad. Sci. USA, 83:3381-3385 (1986). Clements, J. M., et al., "Transcription and processing signals in the 3-phosphoglycerate kinase (PGK) gene from Aspergillus nidulans", Gene, 44:97-105 (1986). Upshall, A., "Filamentous Fungi in Biotechnology", Bio Techniques, 4:158-166 (1986). Penttila, M. E., et al., Cloning of Aspergillus niger genes in yeast. Expression of the gene coding Aspergillus .beta.-glucosidase, Mol. Gen. Genet., 194:494-499 (1984). McKnight, G. L., "Lack of an Aspergillus Promoter Function and Intron Splicing in Saccaromyces", J. Cell. Biochem. Suppl., 9c:137 (1985). Innis, M.A., et al., Expression, Glycosylation, and Secretion of an Aspergillus Glucoamylase by Saccharomyces cerevisiae, Science, 228:21-26 (1985). Briggs, M.S., et al., "Molecular Mechanisms of Protein Secretion: The Role of the Signal Sequence", Advances in Protein Chemistry, 38:109-180 (1986). Nishimori, K., et al., Expression of cloned calf prochymosin gene sequence in Escherichia coli, Gene, 19:337-344 (1982). Beppu, T., "The cloning and expression of chymosin (rennin) genes in microorganisms", Trends in Biotech., 1:85-89 (1983). Emtage, J. S., et al., "Synthesis of calf prochymosin (prorennin) in Escherichia coli", Proc. Natl. Acad. Sci. USA, 80:3671-3675 (1983). Martson, F. A. O., et al., "Purification of Calf Prochymosin (Prorennin) Synthesized in Escherichia coli", Biotechnology 2:800-804 (1984). Kawaguchi, Y., et al., Renaturation and activation of calf prochymosin produced in an insoluble form in Escherichia coli, Journal of Biotechnology, 1:307-315 (1984). Nishimori, K., et al., "Expression of cloned calf prochymosin cDNA under control of the tryptophan promoter", Gene, 29:41-419 (1984). McCaman, M. T., et al., Enzymatic properties and processing of bovine prochymosin synthesized in Escherichia coli, Journal of Biotechnology, 2:177-190 (1985). McGuire, J., "Cloning, Expression, Purification and Testing of Chymosin", from Proceedings of Bio Expo 86 The American Commercial and Industrial Conference and Exposition of Biotechnology, pp. 121-141 (1986). Mellor, J., et al., Efficient synthesis of enzymatically active calf chymosin in Saccaromyces cerevisiae, Gene, 24:1-14 (1983). Goff, C. G., et al., Expression of calf prochymosin in Saccharomyces cerevisiae, Gene, 27:35-46 (1984). Smith, R. A., et al., "Heterologous Protein Secretion from Yeast", Science, 229:1219-1224 (1985). Moir, D. T., et al., Production of Calf Chymosin by the Yeast S. cerevisiae, Developments in Industrial Microbiology, 26:75-85 (1985). Tudzynski, U., et al., "Transformation to senescence with Plasmid Like DNA in the Ascomycete Podospora anserina", Current Genetics 2:181-184 (1980). Freer, S.N., "Fungal Nucleic Acids", In Agricultural Research Service, USDA, Peoria, Illinois, pp. 175-193, (1983). Stahl, U., et al., "Replication and expressin of a bacterial-mitochondrial hybrid plasmid in the fungus Podospora anserina", Proc. Natl. Acad. Sci. USA 79:3641-3645 (1982). Bech, A.-M., et al., "Partial primary structure of Mucor miehei protease", Neth. Milk Dairy 35:275-280 (1981). Moir, D., et al. "Molecular cloning and characterization of double-dtranded cDNA coding for bovine chymosin", Chemical Abstracts, vol. 98, No. 15, p. 173, Abstract No. 120575m (1983). Yelton, M.M., et al., "Transformation of Aspergillus nidulansby using a trpC plasmid", Proc. Natl. Acad. Sci. USA 81:1470-1474 (1984). Ballance, D.J., et al., "Development of a high-frequency transforming vector for Aspergillus nidulans", Gene 36:321-331 (1985). 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| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A transformed Neurospora expression host capable of secreting a heterologous polypeptide, said host being transformed with a vector comprising promoter DNA operably linked to coding DNA, said coding DNA comprising DNA coding for a signal peptide and said heterologous polypeptide. 2. The Neurospora host of claim 1 wherein said promoter DNA comprises a promoter from a gene from a filamentous fungus. 3. The Neurospora host of claim 1 wherein said heterologous polypeptide is biochemically active. 4. The Neurospora host of claim 1 wherein said heterologous polypeptide comprises a mammalian polypeptide. 5. The Neurospora host of claim 4 wherein said mammalian polypeptide comprises chymosin or prochymosin. 6. The Neurospora host of claim 1 wherein said heterologous polypeptide comprises a polypeptide from a filamentous fungus other than said Neurospora host. 7. The Neurospora host of claim 1 wherein said heterologous polypeptide comprises an enzyme. 8. The Neurospora host of claim 1 wherein said enzyme is selected from the group consisting of chymosin, prochymosin, Aspergillus niger glucoamylase, Humicola grisea glucoamylase and Mucor michei carboxyl protease. 9. The Neurospora host of claim 1 wherein said signal peptide is from a polypeptide secreted from a filamentous fungus. 10. The Neurospora host of claim 1 wherein said signal peptide is from a source other than a filamentous fungus. 11. The Neurospora host of claim 10 wherein said 1 signal peptide comprises the signal peptide from a secreted mammalian polypeptide. 12. The Neurospora host of claim 11 wherein said mammalian polypeptide comprises preprochymosin. 13. The Neurospora host of claim 1 wherein said vector further comprises DNA encoding a selection characteristic expressible in said Neurospora hosts. 14. The Neurospora host of claim 13 wherein said selection characteristic is selected from the group consisting acetamidase, pyr4, argB and trpC. 15. The Neurospora host of claim 1 wherein said host comprises Neurospora crassa. 16. A process for producing a heterologous polypeptide comprising: culturing a member of the genera Neurospora transformed with a vector comprising promoter DNA from a fungal gene operably linked to coding DNA, said coding DNA comprising DNA coding for a signal peptide and a heterologous polypeptide wherein said culturing is under conditions which permit the expression of said coding DNA and secretion of said heterologous polypeptide. 17. The process of claim 16 wherein said culturing is carried out in a culture medium comprising utilizable carbon, nitrogen and phosphate sources, surfactant and trace elements. 18. The process of claim 16 further comprising the step of isolating said secreted heterologous polypeptide. 19. The process of claim 16 wherein said promoter is from a fungal gene. 20. The process of claim 19 wherein said fungal gene is from a filamentous fungus. 21. The process of claim 20 wherein said filamentous fungus is selected from the group consisting of Aspergillus, Mucor and Humicola. 22. The process of claim 16 wherein said heterologous polypeptide is biochemically active. 23. The process of claim 16 wherein said heterologous polypeptide comprises a mammalian polypeptide. 24. The process of claim 16 wherein said heterologous polypeptide comprises a polypeptide from a filamentous fungus other than said Neurospora host. 25. The process of claim 16 wherein said heterologous polypeptide comprises an enzyme. 26. The process of claim 16 wherein said signal peptide is from a polypeptide secreted from a filamentous fungus. 27. The process of claim 16 wherein said signal peptide is from a source other than a filamentous fungus. 28. The process of claim 16 wherein said signal peptide comprises the signal peptide from a secreted mammalian polypeptide. 29. The process of claim 16 wherein said vector further comprises DNA encoding a selection characteristic expressible in said Neurospora host. 30. A process for producing a heterologous polypeptide comprising: transforming an Neurospora host, with a vector comprising a DNA construct comprising promoter DNA operably linked to coding DNA, said coding DNA comprising DNA coding for a signal peptide and said heterologous polypeptide, and culturing said transformed filamentous fungus under conditions which permit the expression of said coding DNA and secretion of said heterologous polypeptide. 31. The process of claim 30 wherein said promoter is from a fungal gene. 32. The process of claim 31 wherein said fungal gene is from a filamentous fungus. 33. The process of claim 32 wherein said filamentous fungus is selected from the group consisting of Aspergillus, Mucor and Humicola. 34. The process of claim 30 wherein said heterologous polypeptide is biochemically active. 35. The process of claim 30 wherein said heterologous polypeptide comprises a mammalian polypeptide. 36. The process of claim 30 wherein said heterologous polypeptide comprises a polypeptide from a filamentous fungus other than said Neurospora host. 37. The process of claim 30 wherein said heterologous polypeptide comprises an enzyme. 38. The process of claim 30 wherein said signal peptide is from a polypeptide secreted from a filamentous fungus. 39. The process of claim 30 wherein said signal peptide is from a source other than a filamentous fungus. 40. The process of claim 30 wherein said signal peptide comprises the signal peptide from a secreted mammalian polypeptide. 41. The process of claim 30 wherein said vector further comprises DNA encoding a selection characteristic expressible in said Neurospora host. |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION The present invention is directed to heterologous polypeptides expressed and secreted by filamentous fungi and to vectors and processes for expressing and secreting such polypeptides. More particularly, the invention discloses transformation vectors and processes using the same for expressing and secreting biologically active bovine chymosin and heterologous glucoamylase by a filamentous fungus. BACKGROUND OF THE INVENTION The expression of DNA sequences encoding heterologous polypeptides (i.e., polypeptides not normally expressed and secreted by a host organism) has advanced to a state of considerable sophistication. For example, it has been reported that various DNA sequences encoding pharmacologically desirable polypeptides [e.g., human growth hormone (1), human tissue plasminogen activator (2), various human interferons (6), urokinase (5), Factor VIII (4), and human serum albumin (3)] and industrially important enzymes [e.g., chymosin (7), alpha amylases (8), and alkaline proteases (9)] have been cloned and expressed in a number of different expression hosts. Such expression has been achieved by transforming prokaryotic organisms [e.g., E. coli (10) or B. subtilis (11)] or eukaryotic organisms [e.g., Saccharomyces cerevisiae (7), Kluyveromyces lactis (12) or Chinese Hamster Ovary cells (2)] with DNA sequences encoding the heterologous polypeptide. Some polypeptides, when expressed in heterologous hosts, do not have the same level of biological activity as their naturally produced counterparts when expressed in various host organisms. For example, bovine chymosin has very low biological activity when expressed by E. coli (13) or S. cerevisiae (7). This reduced biological activity in E. coli is not due to the natural inability of E. coli to glycosylate the polypeptide since chymosin is not normally glycosylated (14). Such relative inactivity, both in E. coli and S. cerevisiae, however, appears to be primarily due to improper folding of the polypeptide chain as evidenced by the partial post expression activation of such expressed polypeptides by various procedures. In such procedures, expressed chymosin may be sequentially denatured and renatured in a number of ways to increase biological activity: e.g., treatment with urea (13), exposure to denaturing/renaturing pH (13) and denaturation and cleavage of disulfide bonds followed by renaturation and regeneration of covalent sulfur linkages (15). Such denaturation/renaturation procedures, however, are not highly efficient [e.g., 30% or less recovery of biological activity for rennin (13)], and add considerable time and expense in producing a biologically active polypeptide. Other heterologous polypeptides are preferably expressed in higher eukaryotic hosts (e.g., mammalian cells). Such polypeptides are usually glycopolypeptides which require an expression host which can recognize and glycosylate certain amino acid sequences in the heterologous polypeptide. Such mammalian tissue culture systems, however, often do not secrete large amounts of heterologous polypeptides when compared with microbial systems. Moreover, such systems are technically difficult to maintain and consequently are expensive to operate. Transformation and expression in a filamentous fungus involving complementation of aroD mutants of N. crassa lacking biosynthetic dehydroquinase has been reported (16). Since then, transformation based on complementation of glutamate dehydrogenase deficient N. crassa mutants has also been developed (17). In each case the dehydroquinase (ga2) and glutamate dehydrogenase (am) genes used for complementation were derived from N. crassa and therefore involved homologous expression. Other examples of homologous expression in filamentous fungi include the complementation of the auxotrophic markers trpC, (18) and argB (19) in A. nidulans and the transformation of A. nidulans to acetamide or acrylamide utilization by expression of the A. nidulans gene encoding acetamidase (20). Expression of heterologous polypeptides in filamentous fungi has been limited to the transformation and expression of fungal and bacterial polypeptides. For example, A. nidulans, deficient in orotidine-5'-phosphate decarboxylase, has been transformed with a plasmid containing DNA sequences encoding the pyr4 gene derived from N. crassa (21,32). A. niger has also been transformed to utilize acetamide and acrylamide by expression of the gene encoding acetamidase derived from A. nidulans (22). Examples of heterologous expression of bacterial polypeptides in filamentous fungi include the expression of a bacterial phosphotransferase in N. crassa (23) Dictyostellium discoideum (24) and Cephalosporium acremonium (25). In each of these examples of homologous and heterologous fungal expression, the expressed polypeptides were maintained intracellularly in the filamentous fungi. Accordingly, an object of the invention herein is to provide for the expression and secretion of heterologous polypeptides by and from filamentous fungi including vectors for transforming such fungi and processes for expressing and secreting such heterologous polypeptides. SUMMARY OF THE INVENTION The inventor includes novel vectors for expressing and secreting heterologous polypeptides from filamentous fungi. Such vectors are used in novel processes to express and secrete such heterologous polypeptides. The vectors used for transforming a filamentous fungus to express and secrete a heterologous polypeptide include a DNA sequence encoding a heterologous polypeptide and a DNA sequence encoding a signal sequence which is functional in a secretory system in a given filamentous fungus and which is operably linked to the sequence encoding the heterologous polypeptide. Such signal sequences may be the signal sequence normally associated with the heterologous polypeptides or may be derived from other sources. The vector may also contain DNA sequences encoding a promoter sequence which is functionally recognized by the filamentous fungus and which is operably linked to the DNA sequence encoding the signal sequence. Preferably functional polyadenylation sequences are operably linked to the 3' terminus of the DNA sequence encoding the heterologous polypeptides. Each of the above described vectors are used in novel processes to transform a filamentous fungus wherein the DNA sequences encoding the signal sequence and heterologous polypeptide are expressed. The thus synthesized polypeptide is thereafter secreted from the filamentous fungus. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a restriction map of the Aspergillus niger glucoamylase inserts in pGa1 and pGa5. FIG. 2 depicts the construction of pDJB-gam-1. FIG. 3 depicts the construction of mp19GAPR. FIGS. 4, 5, 6, and 7 depict the construction of pGRG1, pGRG2, pGRG3, and pGRG4. FIGS. 8A-D shows the strategy used to generate mp19 GAPR.sup..DELTA. C1-.sup..DELTA. C4 from mp19 GAPR. FIG. 9 depicts the construction of pCR160. FIG. 10 is a partial restriction map of the Mucor miehei carboxyl protease gene including 5' and 3' flanking sequences. FIGS. 11 A-C is the DNA sequence of Mucor miehei, carboxyl protease including the entire coding sequence and 5' and 3' flanking sequences. FIG. 12 depicts the construction of pMeJB1-7. FIGS. 13 A and B are a partial nucleotide and restriction map of ANS-1. FIG. 14 depicts the construction of pDJB-3. FIG. 15 depicts the construction of plasmid pCJ:GRG1 through pCJ:GRG4. FIG. 16 depicts a restriction endonuclease cleavage map of the 3.7 Kb BamHI fragment from pRSH1. FIG. 17 depicts the construction of pCJ:RSH1 and pCJ:RSH2. FIG. 18 depicts the expression of H. grisea glucoamylase from A. nidulans. DETAILED DESCRIPTION The inventors have demonstrated that heterologous polypeptides from widely divergent sources can be expressed and secreted by filamentous fungi. Specifically, bovine chymosin, glucoamylase from Aspergillus niger and Humicola grises and the carboxyl protease from Mucor miehei have been expressed in and secreted from A. nidulans. In addition, bovine chymosin has been expressed and secreted from A. awamori and Trichoderma reesei. Biologically active chymosin was detected in the culture medium without further treatment. This result was surprising in that the vectors used to transform A. nidulans were constructed to secrete prochymosin which requires exposure to an acidic environment (approximately pH 2) to produce biologically active chymosin. In general, a vector containing DNA sequences encoding functional promoter and terminator sequences (including polyadenylation sequences) are operably linked to DNA sequences encoding various signal sequences and heterologous polypeptides. The thus constructed vectors are used to transform a filamentous fungus. Viable transformants may thereafter be identified by screening for the expression and secretion of the heterologous polypeptide. Alternatively, an expressible selection characteristic may be used to isolate transformants by incorporating DNA sequences encoding the selection characteristic into the transformation vector. Examples of such selection characteristics include resistance to various antibiotics, (e.g., aminoglycosides, benomyl etc.) and sequences encoding genes which complement an auxotrophic defect (e.g. pyr4 complementation of pyr4 deficient A. nidulans, A. awamori or Trichoderma reesei or ArgB complementation of ArgB deficient A. nidulans or A. awamori) or sequences encoding genes which confer a nutritional (e.g., acetamidase) or morphological marker in the expression host. In the preferred embodiments disclosed a DNA sequence encoding the ANS-1 sequence derived from A. nidulans is included in the construction of the transformation vectors of the present invention. This sequence increases the transformation efficiency of the vector. Such sequences, however, are not considered to be absolutely necessary to practice the invention. In addition, certain DNA sequences derived from the bacterial plasmid pBR325 form part of the disclosed transformation vectors. These sequences also are not believed to be necessary for transforming filamentous fungi. These sequences instead provide for bacterial replication of the vectors during vector construction. Other plasmid sequences which may also be used during vector construction include pBR322 (ATCC 37017), RK-2 (ATCC 37125), pMB9 (ATCC 37019) and pSC101 (ATCC 37032). The disclosed preferred embodiments are presented by way of example and are not intended to limit the scope of the invention. DEFINITIONS By "expressing polypeptides" is meant the expression of DNA sequences encoding the polypeptide. "Polypeptides" are polymers of .alpha.-amino acids which are covalently linked through peptide bonds. Polypeptides include low molecular weight polymers as well as high molecular weight polymers more commonly referred to as proteins. In addition, a polypeptide can be a phosphopolypeptide, glycopolypeptide or metallopolypeptide. Further, one or more polymer chains may be combined to form a polypeptide. As used herein a "heterologous polypeptide" is a polypeptide which is not normally expressed and secreted by the filamentous fungus used to express that particular polypeptide. Heterologous polypeptides include polypeptides derived from prokaryotic sources (e.g., .alpha.-amylase from Bacillus species, alkaline protease from Bacillus species, and various hydrolytic enzymes from Pseudomonas, etc.), polypeptides derived from eukaryotic sources (e.g., bovine chymosin, human tissue plasminogen activator, human growth hormone, human interferon, urokinase, human serum albumin, factor VIII etc.), and polypeptides derived from fungal sources other than the expression host (e.g., glucoamylase from A. niger and Humicola grisea expressed in A. nidulans, the carboxyl protease from Mucor miehei expressed in A. nidulans, etc.). Heterologous polypeptides also include hybrid polypeptides which comprise a combination of partial or complete polypeptide sequences derived from at least two different polypeptides each of which may be homologous or heterologous with regard to the fungal expression host. Examples of such hybrid polypeptides include: 1) DNA sequences encoding prochymosin fused to DNA sequences encoding the A. niger glucoamylase signal and pro sequence alone or in conjunction with various amounts of amino-terminal mature glucoamylase codons, and 2) DNA sequences encoding fungal glucoamylase or any fungal carboxy protease, human tissue plasminogen activator or human growth hormone fused to DNA sequences encoding a functional signal sequence alone or in conjunction with various amounts of amino-terminal propeptide condons or mature codons associated with the functional signal. Further, the heterologous polypeptides of the present invention also include: 1) naturally occuring allellic variations that may exist or occur in the sequence of polypeptides derived from the above prokaryotic, eukaryotic and fungal sources as well as those used to form the above hybrid polypeptides, and 2) engineered variations in the above heterologous polypeptides brought about, for example, by way of site specific mutagenesis wherein various deletions, insertions or substitutions of one or more of the amino acids in the heterologous polypeptides are produced. A "biochemically active heterologous polypeptide" is a heterologous polypeptide which is secreted in active form as evidenced by its ability to mediate: 1) the biochemical activity mediated by its naturally occurring counterpart, or 2) in the case of hybrid polypeptides, the biochemical activity mediated by at least one of the naturally occurring counterparts comprising the hybrid polypeptides. Each of the above defined heterologous polypeptides is encoded by a heterologous DNA sequence which contains a stop signal which is recognized by the filamentous fungus in which expression and secretion occurs. When recognized by the host, the stop signal terminates translation of the mRNA encoding the heterologous polypeptide. The "filamentous fungi" of the present invention are eukaryotic microorganisms and include all filamentous forms of the subdivision Eumycotina (26). These fungi are characterized by a vegatative mycelium composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungi of the present invention are morphologically, physiologically, and genetically distinct from yeasts. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as S. cerevisiae is by budding of a unicellular thallus, and carbon catabolism may be fermentative. S cerevisiae has a prominent, very stable diploid phase whereas, diploids exist only briefly prior to meiosis in filamentous fungi like Aspergilli and Neurospora. S. cervisiae has 17 chromosomes as opposed to 8 and 7 for A. nidulans and N. crassa respectively. Recent illustrations of differences between S. cerevisiae and filamentous fungi include the inability of S. cerevisiae to process Aspergillus and Trichoderma introns and the inability to recognize many transcriptional regulators of filamentous fungi (27). Various species of filamentous fungi may be used as expression hosts including the following genera: Aspergillus, Trichoderma, Neurospora, Podospora, Endothia Mucor, Cochiobolus and Pyricularia. Specific expression hosts include A. nidulans (18, 19, 20, 21, 61), A. niger (22), A. awomari, e.g., NRRL 3112, ATCC 22342 (NRRL 3112), ATCC 44733, ATCC 14331 and strain UVK 143f, A. oryzae, e.g., ATCC 11490, N. crassa (16, 17, 23), Trichoderma reesei, e.g. NRRL 15709, ATCC 13631, 56764, 56765, 56466, 56767, and Trichoderma viride, e.g., ATCC 32098 and 32086. As used herein, a "promotor sequence" is a DNA sequence which is recognized by the particular filamentous fungus for expression purposes. It is operably linked to a DNA sequence encoding the above defined polypeptides. Such linkage comprises positioning of the promoter with respect to the initiation codon of the DNA sequence encoding the signal sequence of the disclosed transformation vectors. The promoter sequence contains transcription and translation control sequences which mediate the expression of the signal sequence and heterologous polypeptide. Examples include the promoter from A. niger glucoamylase (39,48), the Mucor miehei carboxyl protease herein, and A. niger .alpha.-glucosidase (28), Trichoderma reesei cellobiohydrolase I (29), A. nidulans trpC (18) and higher eukaryotic promoters such as the SV40 early promoter (24). Likewise a "terminator sequence" is a DNA sequence which is recognized by the expression host to terminate transcription. It is operably linked to the 3' end of the DNA encoding the heterologous polypeptide to be expressed. Examples include the terminator from A. nidulans trpC (18), A. niger glucoamylase (39,48), A. niger .alpha.-glucosidase (28), and the Mucor miehei carboxyl protease herein, although any fungal terminator is likely to be functional in the present invention. A "polyadenylation sequence" is a DNA sequence which when transcribed is recognized by the expression host to add polyadenosine residues to transcribed mRNA. It is operably linked to the 3' end of the DNA encoding the heterologous polypeptide to be expressed. Examples include polyadenylation sequences from A. nidulans trpC (18), A. niger glucoamylase (39,48), A. niger .alpha.-glucosidase (28), and the Mucor miehei carboxyl protease herein. Any fungal polyadenylation sequence, however, is likely to be functional in the present invention. A "signal sequence" is an amino acid sequence which when operably linked to the amino-terminus of a heterologous polypeptide permits the secretion of such heterologus polypeptide from the host filamentous fungus. Such signal sequences may be the signal sequence normally associated with the heterologous polypeptide (i.e., a native signal sequence) or may be derived from other sources (i.e., a foreign signal sequence). Signal sequences are operably linked to a heterologous polypeptide either by utilizing a native signal sequence or by joining a DNA sequence encoding a foreign signal sequence to a DNA sequence encoding the heterologous polypeptide in the proper reading frame to permit translation of the signal sequence and heterologous polypeptide. Signal sequences useful in practicing the present invention include signals derived from bovine preprochymosin (15), A. niger glucoamylase (39), the Mucor miehei carboxyl protease herein and Trichoderma reesei cellulases (29). However, any signal sequence capable of permitting secretion of a heterologous polypeptide is contemplated by the present invention. A "propeptide" or "pro sequence" is an amino acid sequence positioned at the amino terminus of a mature biologically active polypeptide. When so positioned the resultant polypeptide is called a zymogen. Zymogens, generally, are biologically inactive and can be converted to mature active polypeptides by catalytic or autocatalytic cleavage of the propeptide from the zymogen. In one embodiment of the invention a "transformation vector" is a DNA sequence encoding a heterologous polypeptide and a DNA sequence encoding a heterologous or homologous signal sequence operably linked thereto. In addition, a transformation vector may include DNA sequences encoding functional promoter and polyadenylation sequences. Each of the above transformation vectors may also include sequences encoding an expressible selection characteristic as well as sequences which increase the efficiency of fungal transformation. "Transformation" is a process wherein a transformation vector is introduced into a filamentous fungus. The methods of transformation of the present invention have resulted in the stable integration of all or part of the transformation vector into the genome of the filamentous fungus. However, transformation resulting in the maintenance of a self-replicating extra-chromosomal transformation vector is also contemplated. GENERAL METHODS "Digestion" of DNA refers to catalytic cleavage of the DNA with an enzyme that acts only at certain locations in the DNA. Such enzymes are called restriction enzymes, and the sites for which each is specific is called a restriction site. "Partial" digestion refers to incomplete digestion by a restriction enzyme, i.e., conditions are chosen that result in cleavage of some but not all of the sites for a given restriction endonuclease in a DNA substrate. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements as established by the enzyme suppliers were used. In general, about 1 microgram of plasmid or DNA fragment is used with about 1 unit of enzyme and about 20 microliters of buffer solution. Appropriate buffers and substrate amounts with particular restriction enzymes are specified by the manufacturer. Incubation times of about one hour at 37.degree. C. are ordinarily used, but may vary in accordance with the supplier's instructions. After incubation, protein is removed by extraction with phenol and chloroform, and the digested nucleic acid is recovered from the aqueous fraction by precipitation with ethanol. Digestion with a restriction enzyme may be followed by bacterial alkaline phosphatase hydrolysis of the terminal 5' phosphates to prevent the two ends of a DNA fragment from forming a closed loop that would impede insertion of another DNA fragment at the restriction site upon ligation. "Recovery" or "isolation" of a given fragment of DNA from a restriction digest means separation of the digest by a polyacrylamide gel electrophoresis, identification of the fragment of interest, removal of the gel section containing the desired fragment, and separation of the DNA from the gel generally by electroelution (30). "Ligation" refers to the process of forming phosphodiester bonds between two double-stranded nucleic acid fragments (30). Unless otherwise stated, ligation was accomplished using known buffers in conditions with one unit of T4 DNA ligase ("ligase") per 0.5 microgram of approximately equal molar amounts of the DNA fragments to be ligated. "Oligonucleotides" are short length single or double stranded polydeoxynucleotides which were chemically synthesized by the method of Crea et al., (31) and then purified on polyacrylamide gels. "Transformation" means introducing DNA in to an organism so that the DNA is maintained, either as an extrachromosomal element or chromosomal integrant. Unless otherwise stated, the method used herein for transformation of E. coli was the CaCl.sub.2 method (30). A. nidulans strain G191 (University of Glasgow culture collection) was transformed by incubating A. nidulans sphaeroplasts with the transformation vector. The genotype of strain G191 is pabaA1 (requires p-aminobenzoic acid), fwA1 (a color marker), mauA2 (monoamine non-utilizing), and pyrG89 (deficient for orotidine phosphate decarboxlase). Sphaeroplasts were prepared by the cellophane method of Ballance et al. (21) with the following modifications. To digest A. nidulans cell walls, Novozyme 234 (Novo Industries, Denmark) was first partially purified. A 100 to 500 mg sample of Novozyme 234 was dissolved in 2.5 ml of 0.6M KCl. The 2.5 ml aliquot was loaded into a PD10 column (Pharmacia-Upsulla, Sweden) equilibrated with 0.6M KCl. The enzymes were eluted with 3.5 ml of the same buffer. Cellophane discs were incubated in Novozyme 234 (5 mg/ml) for 2 hours, then washed with 0.6M KCl. The digest and washings were combined, filtered through miracloth (Calbiochem-Behring Corp., La Jolla, Calif.), and washed as described (21). Centrifugations were in 50 or 15 ml conical tubes at ca. 1000.times.g for 10 min. Following incubation on ice for 20 min, 2 ml of the polyethylene glycol 4000 solution (250 mg/ml) was added, incubated at room temperature for 5 min. followed by the addition of 4 ml of 0.6M KCl, 50 mM CaCl.sub.2. Transformed protoplasts were centrifuged, resuspended in 0.6M KCl, 50 mM CaCl.sub.2, and plated as described (21). Zero controls comprised protoplasts incubated with 20 .mu.l of 20 mM Tris-HCl, 1 mM EDTA, pH7.4 without plasmid DNA. Positive controls comprised transformation with 5 .mu.g of pDJB3 constructed as described herein. Regeneration frequencies were determined by plating dilutions on minimal media supplemented with 5-10 ppm paba and 500 ppm uridine. Regeneration ranged from 0.5 to 5%. Because of the low transformation frequencies associated with pDJB1, the derivative containing the Mucor acid protease gene (pMeJB1-7) was expected to give extremely low transformation frequencies. Consequently, to obtain pmeJB11-7 transformants of A. nidulans, cotransformation was used. This was accomplished by first constructing a non-selectable vector containing ANS-1, and then transforming sphaeroplasts with a mixture of pmeJB1-7 and the non-selectable vector containing the ANS-1 fragment. The rationale for this approach was that the ANS-1 bearing vector would integrate in multiple copies and provide regions of homology for pMeJB1-7 integration. The ANS-1 vector was prepared by subcloning the PstI-PvuII fragment of ANS-1 (FIGS. 12A and 13B) from pDJB-3 into pUC18 (33). The two plasmids (pMeJB1-7 and the ANS-1 containing vector) were mixed (2.5 .mu.g each) and the above mentioned transformation protocol followed. Transformants obtained with vectors PGRG1-pGRG4 and pDJB-gam were transferred after 3 or 4 days incubation at 37.degree. C. Minimal media agar plates supplemented with 5 ppm p-aminobenzoic acid were centrally inoculated with mycelial transfers from transformants. Three to five days following inoculation of minimal medium plates, spore suspensions were prepared by vortexing a mycelial fragment in 1 ml distilled H.sub.2 O, 0.02% tween-80. Approximately 5.times.10.sup.4 spores were inoculated into 250 ml baffled flasks containing 50 ml of the following medium: (g/l) Maltodextrin M-040 (Grain Processing Corp., Muscatine, Iowa) 50 g, NaNO.sub.3 6 g, MgSO.sub.4.7H20 0.5 g, KCl 0.52 g, KH.sub.2 PO.sub.4, 68 g, 1 ml trace element solution (34), 1 ml MAZU DF-60P antifoam (Mazer Chemicals, Inc., Gurnee, Ill.), 10 ppm p-aminobenzoic acid, and 50 ppm streptomycin sulfate. Alternatives to MAZU, such as bovine serum albumin or other appropriate surfactant may be used. Mucor acid protease secretion was tested in Aspergillus complete medium (20 g dextrose, 1 g peptone, 20 g malt extract per liter). Carbon source regulation of chymosin secretion by Aspergillus nidulans transformants was assessed by measuring secretion in the above-mentioned starch medium relative to the same medium supplemented with 1% fructose, sucrose, or dextrose instead of 5% starch. In all cases, the media were incubated at 37.degree. C. on a rotary shaker (150 rpm). A pDJB3-derived transformant was included as a control. Western blots of the various secreted chymosins and Mucor miehei carboxyl protease were performed according to Towbin, et. al (35). Due to the high concentration of salt in chymosin culture broths and the effect this salt has on gel electrophoresis a desalting step was necessary. Pre-poured G-25 columns (Pharmacia, PD10) were equilibriated with 50 mM Na.sub.2 HPO.sub.4, pH 6.0. A 2.5 ml aliquot of culture broth was applied to the column. The protein was eluted with 3.5 ml of the same buffer. The heterologous polypeptides present on the blots were detected by contacting the nitrocellulose blots first with rabbit anti-chymosin (36) or rabbit anti-Mucor miehei carboxy protease serum (36). The blots were next contacted with goat-anti-rabbit serum conjugated with horseradish peroxidase (Bio-Rad, Richmond, Calif.) and developed. Prior to loading on the gels, 50 .mu.l of medium (desalted in the case of chymosin) was mixed with 25 .mu.l of SDS sample buffer. .beta.-mercaptoethanol was added to a final concetration of 1%. The sample was heated in a 95.degree. C. bath for 5 minutes after which 40-50 .mu.l of sample was loaded on the gel. Each gel was also loaded with 2 .mu.l each of 650, 65 and 6.5 .mu.g/ml chymosin standards and molecular weight markers. Western blots of pmeDJ1-7 transformants were similarly analyzed except that gel permeation was not performed. Protease activity was detected as described by Sokol, et. al. (37). Luria broth was supplemented with 1-1.5% skim milk (Difco) and 30-35 ml was poured into a 150 mm petri dish. An aliquot of 2 to 5 .mu.l of culture medium was spotted on the plate. The plate was incubated over night at 37.degree. C. in a humidity box. The activity was determined based on the amount of milk clotting occurring on the plate measured in mm. The plates were co-spotted with dilutions of 100 CHU/ml or 16.6 CHU/ml rennin (CHU-Chr Hansen Unit, Chr Hansen's Laboratorium, A./S., Copenhagen). The relationship between the diameter of the coagulation zone (mm) and the centration of enzyme is logarithmic. In order to distinguish between types of proteases, pepstatin, an inhibitor of the chymosin type of carboxyl protease, was used to inhibit protease activity attributable to chymosin. Samples of chymosin mutants and control broths were preincubated with a 1:100 dilution of 10 mM pepstatin in DMSO for 5 minutes before analyzing for protease activity. Glucoamylase secretion by pDJB-gam-1 transformants in 5% starch media was assessed using an assay based on the ability of glucoamylase to catalyze the conversion of p-nitrophenol-a-glucopyranoside (PNPAG) (38) to free glucose and p-nitrophenoxide. The substrate, PNPAG, was dissolved in DMSO at 150 mg/ml and 3 to 15 .mu.l aliquots were diluted to 200 ul with 0.2 M sodium acetate, 1 mM calcium chloride at pH 4.3. A 25 .mu.l sample was placed into a microtitre plate well. An equal volume of standards ranging from 0 to 10 Sigma A. niger units/ml (Sigma Chemical Co., St. Louis, Mo.) were placed in separate wells. To each well, 200 .mu.l of PNPAG solution at 2.25 to 11.25 mg/ml was added. The reaction was allowed to proceed at 60.degree. C. for 0.5 to 1 hour. The time depended upon the concentration of enzyme. The reaction was terminated by the addition of 50 .mu.l of 2 M trizma base. The plate was read at 405 nm. The concentration of enzyme was calculated from a standard curve. Unless otherwise stated, chromosomal DNA was extracted from filamentous fungi by the following procedure. The filamentous fungus was grown in an appropriate medium broth for 3 to 4 days. Mycelia were harvested by filtering the culture through fine cheesecloth. The mycelia were rinsed thoroughly in a buffer of 50 mM tris-HCl, pH7.5, 5 mM EDTA. Excess liquid was removed by squeezing the mycelia in the cheesecloth. About 3 to 5 grams of wet mycelia were combined with an equivalent amount of sterile, acid-washed sand in a mortar and pestle. The mixture was ground for five minutes to form a fine paste. The mixture was ground for another five minutes after adding 10 ml of 50 mM tris-HCl, pH 7.5, 5 mM EDTA. The slurry was poured into a 50 ml capped centrifuge tube and extracted with 25 ml of phenol-chloroform (equilibrated with an equal volume of 50 mM tris-HCl, pH 7.5, 5 mM EDTA). The phases were separated by low speed centrifugation. The aqueous phase was saved and reextracted three times. The aqueous phases were combined (about 20 ml total volume) and mixed with 2 ml of 3 M sodium acetate, pH 5.4 in sterile centrifuge tubes. Ice cold isopropanol (25 ml) was added and the tubes were placed at -20.degree. C. for one hour. The tubes were then centrifuged at high speed to pellet the nucleic acids, and the supernatant fluid was discarded. Pellets were allowed to air dry for 30 minutes before resuspending in 400 .mu.l of 10 mM tris-HCl, pH 7.5, 1 mM EDTA (TE buffer). Pancreatic ribonuclease (Sigma Chemical Co., St. Louis, Mo.) was added to a final concentration of 10 .mu.g per ml, and the tubes were incubated for 30 minutes at room temperature (30). Ribonuclease was then removed by extraction with phenol-chloroform. The aqueous layer was carefully removed and placed in a tube which contained 40 .mu.l of 3M sodium acetate, pH 5.4. Ice cold ethanol was layered into the solution. The DNA precipitated at the interface and was spooled onto a glass rod. This DNA was dried and resuspended in a small volume (100 to 200 .mu.l) of TE buffer. The concentration of DNA was determined spectrophotometrically at 260 nm (30). To confirm the chromosomal integration of chymosin DNA sequences in selected transformants Southern hybridizations were performed (30). Spore suspensions of transformants were inoculated into Aspergillus complete medium and incubated at 37.degree. C. on a rotary shaker for 24-48 hrs. The medium was non-selective in that it was supplemented with 5 ppm p-aminobenzoic acid and contained sufficient uracil for growth of the auxotrophic parent. In effect, these Southerns also tested for the stability of the transformants. The mycelium was filtered, ground in sand, and the DNA purified as previously described. Transformant DNA was then digested with various restriction enzymes and fragments separated by agarose gel electrophoresis. Control lanes included digested pDJB3 transformant DNA and undigested DNA. Gels were stained with ethidium bromide, photographed, blotted to nitrocellulose or nytran (Schleicher and Schuell, Keene, N.H.), and probed with radiolabeled plasmids or specific fragments. |
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