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(12) United States Patent (10) Patent No.: US 6,365,390 B1 Blum Et Al

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(12) United States Patent (10) Patent No.: US 6,365,390 B1 Blum Et Al USOO6365390B1 (12) United States Patent (10) Patent No.: US 6,365,390 B1 Blum et al. (45) Date of Patent: Apr. 2, 2002 (54) PHENOLIC ACID ESTERASES, CODING Chen et al. (1995) “A Cyclophilin from the Polycentric SEQUENCES AND METHODS Anaerobic Rumen Fungus Orpinomyces sp. Strain PC-2 is Highly Homologous to Vertebrate Cyclophilin B” Proc. (75) Inventors: David L. Blum, San Diego, CA (US); Natl. Acad. Sci. USA 92:2587-2591.* Irina Kataeva, Athens, GA (US); Chritov and Prior (1993) “Esterases of Xylan-Degrading Xin-Liang Li, Athens, GA (US); Lars Microorganisms: Production, Properties, and Significance' G. Ljungdahl, Athens, GA (US) Enzyme Microb. Technol. 15:460-475.* Dalrymple and Swadling (1997) “Expression of a Butyriv (73) Assignee: University of Georgia Research ibrio fibrisolvens E14 Gene (cinB) Encoding an Enzyme Foundation, Inc., Athens, GA (US) with Cinnamoyl Ester Hydrolase Activity is Negatively Regulated by the Product of an Adjacent Gene (cinR)” (*) Notice: Subject to any disclaimer, the term of this Microbiology 143:1203–1210. patent is extended or adjusted under 35 Dalrymple et al. (1996) “Cloning of a Gene Encoding U.S.C. 154(b) by 0 days. Cinnamoyl Ester Hydrolase from the Ruminal Bacterium Butyrivibrio fibrisolvens E14 by a Novel Method” FEMS (21) Appl. No.: 09/390,234 Microbiology Letters 143:115-120. De Vries et al. (1997) “The faeA Genes from Aspergillus (22) Filed: Sep. 3, 1999 niger and Aspergillus tubingensis Encode Ferulic Acid Related U.S. Application Data Esterases Involved in Degradation of Complex Cell Wall (60) Provisional application No. 60/099,136, filed on Sep. 4, Polysaccharides' Applied and Environmental Microbiology 1998. 63:46.38-4644. Faulds and Williamson (1991) “The Purification and Char acterization of 4-Hydroxy-3-Methoxycinnamic (Ferulic) (51) Int. Cl. ............................ C12N 9/18; CO7H 21/04 Acid Esterase from Streptomyces Olivochromogenes' Jour (52) U.S. Cl. .................... 435/197; 435/183; 435/320.1; nal of General Microbiology 137:2339–2345. 435/252.3; 536/23.1; 536/23.2; 530/350 Felix and Ljungdahl (1993) “The Cellulosome: The Exo (58) Field of Search ................................. 435/197, 183, cellular Organelle of Clostridium” Annu. Rev. Microbiol. 435/320.1, 252.3; 536/23.1, 23.2; 530/350 47:791-819. Ferreira et al. (1993) “A Modular Esterase from Pseudomo (56) References Cited nas fluorescens subsp. cellulosa Contains a Non-Catalytic U.S. PATENT DOCUMENTS Cellulose-Binding Domain Biochemical Journal 2.94:349-355. 5,882,905 A 3/1999 Saha et al. .................. 435/105 Flint et al. (193) “A Bifunctional Enzyme, with Separate FOREIGN PATENT DOCUMENTS Xylanase and f(1,3-1,4)-Glucanase Domains, Encoded by the XnyD Gene of Ruminococcus flavefaciens' Journal of EP O513140 B1 9/1995 ............ D21C/9/10 Bacteriology 175:2943-2951. GB 23.01.103 11/1996 ............ C12N/9/18 Fontes et al. (1995) “Evidence for a General Role for WO 98/46768 10/1998 ........... C12N/15/55 Non-Catalytic Thermostabilizing Domains in Xylanases from Thermophilic Bacteria" Biochem. J. 307:151-158. OTHER PUBLICATIONS Genbank Accession No. AF047761, CloStridium thermocel lum Xylanase V and U. Blum et al. (1999) “Characterization of a Feruloyl Esterase Genbank Accession No. L48074, Aspergillus fumigatus from the Anaerobic Fungus Orpinomyces sp. Strain PC-2” dipeptidyl peptidase. Abstracts. 99" General Meeting of the American Society for Microbiology. Chicago, IL. May 30-Jun. 3, 1999. vol. 99, (List continued on next page.) pp. 430-431. Borneman and Akin (1990) “Lignocellulose Degradation by Primary Examiner Rebecca E. Prouty Rumen Fungi and Bacteria: Ultrastructure and Cell Wall ASSistant Examiner-Richard Hudson Degrading Enzymes' In: Microbial and Plan Opportunities (74) Attorney, Agent, or Firm-Greenlee Winner and to Improve Lignocellulose Utilization by Ruminants. D.E. Sullivan PC Akin; L.G. Ljungdahl; J.R. Wilson; and P.J. Harris (Eds.) (57) ABSTRACT Elsevier Science Publishing Co. New York, NY. pp. 325-339.* Described herein are four phenolic acid esterases, three of Borneman et al. (1992) “Purification and Partial Character which correspond to domains of previously unknown func ization of Two Feruloyl Esterases from the Anaerobic Fun tion within bacterial xylanases, from XynY and XynZ of gus Neocallimastix Strain MC-2' Applied and Environmen Clostridium thermocellum and from a Xylanase of Rumino tal Microbiology 58:3762–3766.* coccus. The fourth Specifically exemplified Xylanase is a Borneman et al. (1990) “Assay for trans-p-Coumaroyl protein encoded within the genome of Orpinomyces PC-2. Esterase. Using a Specific Substrate from Plant Cell Walls” The amino acids of these polypeptides and nucleotide Analytical Biochemistry 190:129-133.* Sequences encoding them are provided. Recombinant host Castanares and Wood (1992) “Purification and Character cells, expression vectors and methods for the recombinant ization of a Feruloyl/p-Coumaroyl Esterase from Solid production of phenolic acid esterases are also provided. State Cultures of the Aerobic Fungus Penicillium pinophi lum” Biochemical Society Transactions 20:275S.* 26 Claims, 11 Drawing Sheets US 6,365,390 B1 Page 2 OTHER PUBLICATIONS McDermid et al. (1990) “Esterase Activities of Fibrobacter Succinogenes subsp. Succinogenes S85” Applied and Envi Genbank Accession No. M22624, CloStridium thermocel ronmental Microbiology 56:127-132. lum Xylanase Z. McSweeney et al. (1998) “Butyrivibrio spp. and Other Genbank Accession No. P31471, Escherichia coli 44.1 kD Xylanolytic Microorganisms from the Rumen have Cin protein. namoyl Esterase Activity” Anaerobe 4:57-65. Genbank Accession No. P51584, CloStridium thermocellum Ralph et al. (1995) “Lignin-Ferulate Cross-Links in Xylanase Y. Grasses: Active Incorporation of Ferulate Polysaccharide Genbank Accession No. S58235, Ruminococcus sp. Xyla Esters Into Ryegrass Lignins' Carbohydrate Research Sc. 2.75:167-178. Genbank Accession No. X83269, CloStridium thermocellum Sakka et al. (1996) “Identification and Characterization of Xylanase Y. Cellulose-Binding Domains in Xylanase A of CloStridium Grépinet et al. (1988) “Nucleotide Sequence and Deletion Stercorarium ' Ann. NYAcad. Sci. 782:241-251. Analysis of the Xylanase Gene (xynZ) of Clostridium ther Sakka et al. (1993)“Nucleotide Sequence of the Clostridium mocellum' Journal of Bacteriology 170:4582–4588. Stercorarium XynA Gene Encoding Xylanase A: Identifica Kirby et al. (1998) “Plant Cell Wall Degrading Enzyme tion of Catalytic and Cellulose Binding Domains' BioSci. Complexes from the Cellulolytic Rumen Bacterium Rumi Biotech. Biochen. 57:273-277. nococcus flavefaciens' Biochemical Society Transactions Arakaki et al. Xylanase 1 from RuminococcuS Sp. With a 26:S169. new pattern of domain shuffling, Genbank Accession No.: MacKenzie and Bilous (1988) “Ferulic Acid Esterase Activ ity from Schizophyllum commune' Applied and Environ Z49970, Aug. 1995. mental Microbiology 54:1170–1173. * cited by examiner U.S. Patent Apr. 2, 2002 Sheet 2 of 11 US 6,365,390 B1 XUÁX ZuÁX U.S. Patent Apr. 2, 2002 Sheet 3 of 11 US 6,365,390 B1 Molecular mass (kDa) -o-o-o-o--m----0 2000 232 43 13.7 120 O3O 1 OO Protein H 0.25 -O- FAE activity O.20 0. 15 O 1 O 0.05 0.00 O 5 1 O 15 2O Elution volume (ml) F.G. 3 U.S. Patent Apr. 2, 2002 Sheet S of 11 US 6,365,390 B1 U.S. Patent Apr. 2, 2002 Sheet 6 of 11 US 6,365,390 B1 1OO 8 O 6 O F.G. 6A 4. O 2 O O 1 O 20 30 40 50 60 7O 80 90 100 Temperature (C) 1OO 8 O 6 O FIG. 6B 4 O e 2 O O - O 2 4 6 8 1O 12 U.S. Patent Apr. 2, 2002 Sheet 7 of 11 US 6,365,390 B1 FIG. 7 U.S. Patent Apr. 2, 2002 Sheet 8 of 11 US 6,365,390 B1 co2 So S 3 s 3 g g g & 2 (%) AAgow empere o o d d s of C OO in a s O als e w N o ve 2 3 3 S S 3 So 3do so o2 o q- was (9) AAgoe eagee U.S. Patent Apr. 2, 2002 Sheet 10 of 11 US 6,365,390 B1 N S Signal peptide FAE domain Unknown domain FIG. 10 U.S. Patent Apr. 2, 2002 Sheet 11 of 11 US 6,365,390 B1 0.09 0.08 o 0.06 0.05 0.04 O.O3. 0.02 0.01 OOO US 6,365,390 B1 1 2 PHENOLIC ACID ESTERASES, CODING There is a need in the art for phenolic acid esterases, SEQUENCES AND METHODS feruloyl esterases and/or coumaroyl esterases in pure form which degrade plant cell wall materials, and certain other CROSS REFERENCE TO RELATED Substrates, and for DNA encoding these enzymes to enable APPLICATIONS methods of producing ferulic acid and/or coumaric acid as This application claims priority from United States Pro well as facilitating degradation of plant cell wall materials. visional Application No. 60/099,136, filed Sep. 4, 1998. SUMMARY OF THE INVENTION ACKNOWLEDGEMENT OF FEDERAL The present invention provides novel phenolic acid RESEARCHSUPPORT 1O esterases, having feruloyl esterase and coumaroyl esterase This invention was made, at least in part, with finding activities, and coding Sequences for Same. from the United States Department of Energy (Grant No. One phenolic acid esterase of the present invention cor DE-FG05 93ER 20127). Accordingly, the United States responds to a domain of previously unknown function from Government has certain rights in this invention. Xylanase Y of Clostridium thermocellum. The recombi 15 nantly expressed domain polypeptide is active and has an BACKGROUND OF THE INVENTION amino acid sequence as given in FIG. 1 as “XynY Clotm.” The field of the present invention is the area of enzymes The nucleotide Sequence encoding the esterase polypeptide which degrade plant cell walls, and certain other Substrates, is given in Table 5, nucleotides 2383-3219, exclusive of in particular, the phenolic acid esterases, feruloyl esterases translation start and stop signals.
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