USOO598.1835A United States Patent (19) 11 Patent Number: 5,981,835 Austin-Phillips et al. (45) Date of Patent: Nov. 9, 1999 54) TRANSGENIC PLANTS AS AN Brown and Atanassov (1985), Role of genetic background in ALTERNATIVE SOURCE OF Somatic embryogenesis in Medicago. Plant Cell Tissue LIGNOCELLULOSC-DEGRADING Organ Culture 4:107-114. ENZYMES Carrer et al. (1993), Kanamycin resistance as a Selectable marker for plastid transformation in tobacco. Mol. Gen. 75 Inventors: Sandra Austin-Phillips; Richard R. Genet. 241:49-56. Burgess, both of Madison; Thomas L. Castillo et al. (1994), Rapid production of fertile transgenic German, Hollandale; Thomas plants of Rye. Bio/Technology 12:1366–1371. Ziegelhoffer, Madison, all of Wis. Comai et al. (1990), Novel and useful properties of a chimeric plant promoter combining CaMV 35S and MAS 73 Assignee: Wisconsin Alumni Research elements. Plant Mol. Biol. 15:373-381. Foundation, Madison, Wis. Coughlan, M.P. (1988), Staining Techniques for the Detec tion of the Individual Components of Cellulolytic Enzyme 21 Appl. No.: 08/883,495 Systems. Methods in Enzymology 160:135-144. de Castro Silva Filho et al. (1996), Mitochondrial and 22 Filed: Jun. 26, 1997 chloroplast targeting Sequences in tandem modify protein import specificity in plant organelles. Plant Mol. Biol. Related U.S. Application Data 30:769-78O. 60 Provisional application No. 60/028,718, Oct. 17, 1996. Divne et al. (1994), The three-dimensional crystal structure 51 Int. Cl. ............................. C12N 15/82; C12N 5/04; of the catalytic core of cellobiohydrolase I from Tricho AO1H 5/00 derma reesei. Science 265:524-528. 52 U.S. Cl. ..................... 800/278; 536/23.1; 536/23.74; Ghangas & Wilson (1988), Cloning of the Thermomono 435/69.1; 435/70.1; 435/468; 435/469; Spora fusca endoglucanase E2 gene in Streptomyces liv 435/410; 435/414; 800/278; 800/284; 800/317.3 idans: Affinity purification and functional domains of the 58 Field of Search ................................ 536/23.74, 23.1; cloned gene product. Appl. Envir. Microbiol. 54:2521-2526. 800/205, DIG. 24, DIG. 43, 278, 284,317.3; Grohmann et al. (1992), Potential for fuels from biomass 435/69.1, 70.1, 468, 469, 410, 414 and wastes. In: Emerging technologies for materials and chemicals from biomass, Eds. R.M. Powell, T.P. Schultz and 56) References Cited R. Narayan. ACS symposium series 576. Henrissat et al. (1995), Synergism of cellulases from Tri U.S. PATENT DOCUMENTS choderma reesei in the degradation of cellulose. Bio/Tech 5,432,074 7/1995 Evans et al.. nology 3:722-726. 5,457.046 10/1995 Wöldike et al.. Horsh et al. (1985), A simple and general method for 5,529,919 6/1996 Knowles et al.. transferring genes into plants. Science 227:1229-1231. Irwin et al. (1993), Activity studies of eight purified cellu FOREIGN PATENT DOCUMENTS lases: Specificity, Synergism, and binding domain effects. WO 87/0O865 2/1987 WIPO. Biotechnol. Bioeng. 42:1002-1013. WO 94/26880 11/1994 WIPO. Ishida et al. (1996), High efficiency transformation of maize WO 94/2946O 12/1994 WIPO. mediated by Agrobacterium tumefaciens. Nature Biotech nology 14:745-750. OTHER PUBLICATIONS Keegstra et al. (1993), Targeting of proteins into chloro Adney et al. (1994), Cellulase assays. In: Enzymatic con plasts. Physiologia Plantarum 93:157-162. version of biomass for fuels production, Eds. M. E. Himmel, Lao et al. (1991), J. Bacteriol. 173:3397–3407. J.O. Baker & R.P. Overend. ACS symposium series 566. Mason et al. (1988), Proteins homologous to leaf glycopro Aspegren et al. (1995), Secretion of a heat-stable fungal teins are abundant in Stems of dark-grown Soybean Seed B-glucanase from transgenic, Suspension-cultured barley lings. Analysis of proteins and cDNAs. Plant Mol. Biol. cells. Molecular Breeding 1:91-99. 11:845-856. Baker et al. (1992), Thermal denaturation of T. reesei McBride and Summerfelt (1990), Improved binary vectors cellulases Studied by differential Scanning calorimetry and for Agrobacterium mediated plant transformation. Plant tryptophan fluorescene. Apply. Biochem. BiophyS. MOI. Biol. 14:269-276. 34:217-231. (List continued on next page.) Bednarek (1991), The barley lectin carboxy-terminal pep tide is a vacuolar protein Sorting determinant in plants. The Primary Examiner-Gary BenZion Plant Cell 3:1195-1206. ASSistant Examiner-Ousama Zaghmout Belkacemi et al. (1996), Enzymatic hydrolysis of timothy Attorney, Agent, or Firm-DeWitt Ross & Stevens S. C. grass pretreated by ammonia fiber explosion. In: Liquid fuels 57 ABSTRACT and industrial products from renewable resources, Proceed ings of the third liquid fuel conference, Eds. J.S. Cundiff, Transgenic plants which express cellulose-degrading E.E. Gavett, C. Hansen, C. Peterson, M.A. Sanderson, H. enzymes, methods to make the transgenic plants, and meth Shapouri & D.L. VanDyne. ASAE publication 08-96 pp. ods to use the cellulose-degrading enzymes produced by the 232-240. transgenic plants are disclosed. Bingham et al. (1975), Breeding alfalfa which regenerates from callus tissue in culture. CropSci. 15:719-721. 17 Claims, 4 Drawing Sheets 5,981,835 Page 2 OTHER PUBLICATIONS Zambryski, P., J. Tempe, and J. Schell (1989), Transfer and function of T-DNA genes from Agrobacterium Ti and Ri McBride et al. (1994), Controlled expression of plastid plasmids in plants. Cell 56: 193-201. transgenes in plants based on a nuclear DNA-encoded and plastid-targeted T7 RNA polymerase. Proc. Natl. Acad. Sci. Zhang et al. (1995), Characterization of a Thermomono USA 91:7301-73.05. spora fusca exocellulase. Biochemistry 34:3386-3395. Micelli et al. (1996), Integrated treatments of steam explo Xu et al. FEBS Letters. 1994. Vol. 348: 206-210. Sion and enzymatic hydrolysis to produce energetic and Napoli et al. The Plant Cell. 1989. Vol. 2: 278-289. industrial products from lignocellulosic biomasses. Agro food Industry Hi-tech 7:25-28. Carvalho et al. The EMBO Journal. 1992. vol. 11: Murashige and Skoog (1962), A revised medium for rapid 2595-26O2. growth and bioassays with tobacco tissue cultures. Physiol. Somero, George N., Proteins and Temperature, Annu. Rev. Plf 15:473-497. Physiol. (1995), 57:43–68. Pentilla et al. (1987), Yeast 3:175-185. Jacobs et al., Post-transcriptional B-1,3-glucanase gene Sonnewaldet. al. (1991), Transgenic tobacco plants express Silencing involves increased transcript turnover that is trans ing yeast-derived invertase in either the cytosol, vacuole or lation-independent, The Plant Journal (1977), apoplast: a powerful tool for Studying Sucrose metabolism 12(4):885–893. and sink/Source interactions. The Plant J. 1:95-106. Spezio et al. (1993), Crystal structure of the catalytic domain Davies et al., Genetics of homology-dependent gene Silenc of a thermophilic endocellulase. Biochemistry ing in Arabidopsis; a role for methylation, The Plant Journal 32.9906-9916. (1997), 12(4): 791–804. Tucker et al. (1989), Ultra-thermostable cellulases from De Loose et al., Primary Structure of a homonally egulated Acidothermus cellulolyticuS comparison of temperature f-glucanase of Nicotiana plumbaginifolia, Gene (1988), optima with previously reported cellulases. Biotechnology 70:13-23. 7:817-82O. Castresana et al., Tissue-Specific and Pathogen-Induced Vasil et al. (1993), Rapid production of transgenic wheat Regulation of a Nicotiana plumbaginifolia B-1,3-Glucanase plants by direct particle bombardment of cultured immature embyros. Bio/Technology 11:1553–1558. Gene, The Plant Cell (1990), 2:1131-1143. Wandelt et al. (1992), Vicilin with carboxy-terminal KDEL Smyth, David R., Gene Silencing: CoSuppression at a dis is retained in the endoplasmic reticulum and accumulates to tance, Current Biology (1997), vol. 7, No. 12, R793–R795. high levels in the leaves of transgenic plants. Plant J. Meyer, Peter, Understanding and controlling transgene 2:181-192. expression, Tibtech (Sep. 1995), 13:332–337. U.S. Patent Nov. 9, 1999 Sheet 1 of 4 5,981,835 <No.,No..No.D>TEDETT|de=svw U.S. Patent Nov. 9, 1999 Sheet 2 of 4 5,981,835 U.S. Patent Nov. 9, 1999 Sheet 3 of 4 5,981,835 U.S. Patent Nov. 9, 1999 Sheet 4 of 4 5,981,835 5,981,835 1 2 TRANSGENIC PLANTS AS AN enzymes which catalyze the cleavage of cellulosic or ligno ALTERNATIVE SOURCE OF cellulosic materials. Explicitly, but not eXclusively, included LIGNOCELLULOSC-DEGRADING within this definition are those cellulases which fall under ENZYMES the Enzyme Classification heading EC 3.2.1.x. Various genes encoding cellulases have also been isolated and char Priority is claimed to provisional application Serial No. acterized. 60/028,718, filed Oct. 17, 1996. For instance, genes which encode endoglucanases from This invention was made with United States government the fungus Trichoderma reesei are known and have been Support awarded by the following agencies: Successfully incorporated and expressed in yeast. See, for DOE Grant No. DE-FCO5-92OR22072; instance, Pentilla et al. (1987). Likewise, cellulase E2 (EC DOE Grant No. DE-FC05-92OR22072 (via CPBR, Ref. 3.2.1.4) and cellulase E3 (EC 3.2.1.91) from the thermo No. OR22072-34; tolerant bacterium Thermomonospora fusca are known. See USDA Grant NOS. 94-34190-1204 and 92-34190-6941 Lao et al. (1991), Spezio et al. (1993) and Zhang et al. (via Purdue, Award Nos. 5930120-28 and 5930213-17). (1995). The United States has certain rights in this invention. 15 From a functional viewpoint, cellulases are catagorized into two large Sub-groups based upon whether they catalyze FIELD OF THE INVENTION cleavage from the cellulose chain ends (exocellulases) or if The present invention is directed to the production of they catalyze cleavage in the middle of the cellulose chain cellulose-degrading enzymes in genetically recombinant (endocellulases). For instance, cellobiohydrolase I of T. plants and the recombinant plants themselves.