US008129590B2 (12) United States Patent (10) Patent No.: US 8,129,590 B2 Maranta et al. (45) Date of Patent: Mar. 6, 2012 (54) POLYPEPTIDES HAVING ACETYLXYLAN (56) References Cited ESTERASE ACTIVITY AND POLYNUCLEOTDESENCODING SAME U.S. PATENT DOCUMENTS 5,681,732 A 10, 1997 De Graaffet al. (75) Inventors: Michelle Maranta, Davis, CA (US); 5,763,260 A * 6/1998 De Graaffet al. ............ 435,274 Kimberly Brown, Elk Grove, CA (US) FOREIGN PATENT DOCUMENTS EP O 507 369 3, 1992 (73) Assignee: Novozymes A/S, Bagsvaerd (DK) JP 2001054383. A 2, 2001 WO WO 2005/OO 1036 1, 2005 (*) Notice: Subject to any disclaimer, the term of this WO 2009073709 A1 6, 2009 patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 196 days. Margolles-Clark et al., Accetyl Xylan esterase from Trichoderma reesei contains an active-site serine residue and a cellulose-binding (21) Appl. No.: 12/327,347 domain, 1996, Eur: J. Biochem. 237: 553-560. Henrissat B. A classification of glycosylhydrolases based on amino Filed: Dec. 3, 2008 acid sequence similarities, 1991, Biochem. J. 280: 309-316. (22) Henrissat et al., Updating the sequence-based classification of glycosyl hydrolases, 1996, Biochem. J. 316: 695-696. (65) Prior Publication Data Sundberg et al., 1991, Biotechnol. Appl. Biochem. 13: 1-11. US 201O/OO43105 A1 Feb. 18, 2010 Sundberg et al. Purification and Properties of Two Acetylxylan Esterases of Trichoderma reesei, 1991, Biotechnol. Appl. Biochem. 13: 1-11. Related U.S. Application Data Written Opinion of the International Searching Authority for PCT/ (60) Provisional application No. 60/992,995, filed on Dec. US2008/085.380. 6, 2007. * cited by examiner Primary Examiner — Brent T Page (51) Int. C. (74) Attorney, Agent, or Firm — Robert L. Starnes; Eric J. CI2N 15/31 (2006.01) Fechter CI2N 15/82 (2006.01) CI2N IS/10 (2006.01) (57) ABSTRACT CI2N 15/52 (2006.01) The present invention relates to isolated polypeptides having AOIH 5/00 (2006.01) acetylxylan esterase activity and isolated polynucleotides (52) U.S. C. ........ 800/288: 800/284; 800/295: 800/278: encoding the polypeptides. The invention also relates to 435/320.1; 435/468; 536/23.1:536/23.2: nucleic acid constructs, vectors, and host cells comprising the 536/23.7 polynucleotides as well as methods of producing and using (58) Field of Classification Search ........................ None the polypeptides. See application file for complete search history. 27 Claims, 4 Drawing Sheets U.S. Patent Mar. 6, 2012 Sheet 1 of 4 US 8,129,590 B2 JASÐVÌMÔXIIJ,EN3 LIZ G09 L0.9 IZL 998 U.S. Patent Mar. 6, 2012 Sheet 2 of 4 US 8,129,590 B2 g|-61-I 600I T80I GZZI U.S. Patent Mar. 6, 2012 Sheet 3 of 4 US 8,129,590 B2 Pst (7918) Na2Tpipromoter Bcil (7802) Bcli (7509) EcoRI (7494) Pst (7404) Bcf. (7288) s Aval (430) Pna2 minus TATA box ins AXE2 Bcli (6995 Aval (760) Sali (6980) NXmal (1125) Pmei (6975) Aval (1125) Apa Li (6297) Bell (1356) AMG Terminator WEcoRV (1440) coRV (1573) Clai (1956) Bcli (2014) Saci (2187) -Lactamase EcoRV (2266) Apa Li (2737) Apa L (5051) EcoRV (2856) ANDS Apa LI (4554) Pst (3498) Pnei (4328) Bam H. (3536) Pst (4072) mal (3780) Aval (3780) Smal (3782) Pst (3886) Fig. 2 U.S. Patent Mar. 6, 2012 Sheet 4 of 4 US 8,129,590 B2 HindIII (235) Kpni (245) Saci (251) Apa LI (4790 am Hl (253) CoE1 or EcoRI (284) st (690) Aval (736) Aval (1066) pHinsAXE2 5228 bp Xmal (1431) bla Aval (1431) Small (1433) SSEcoRI (1599) Apa L (3544) VPst (1608) EcoRV (1611) Not (1626) Ava (1632) Nicol (3177) Xbal (1644) kan f1 Ori Psti (2798) BCII (2586) Fig. 3 US 8,129,590 B2 1. 2 POLYPEPTDES HAVING ACETYLXYLAN Margolles-Clark et al., 1996, Eur: J. Biochem. 237: 553 ESTERASE ACTIVITY AND 560, disclose an acetylxylan esterase from Trichoderma POLYNUCLEOTDESENCODING SAME reesei. Sundberg and Poutanen, 1991, Biotechnol. Appl. Bio chem. 13: 1-11, disclose the purification and properties of two CROSS-REFERENCE TO RELATED acetylxylan esterases of Trichoderma reesei. WO 2005/ APPLICATION 001036 discloses an acetylxylan esterase gene from Tricho derma reesei. U.S. Pat. No. 5,681,732 discloses an acetylxy This application claims the benefit of U.S. Provisional lan esterase gene from Aspergillus niger. U.S. Pat. No. 5,763, Application No. 60/992,995, filed Dec. 6, 2007, which appli 260 discloses methods for altering the properties of cation is incorporated herein by reference. 10 acetylated Xylan. The present invention relates to polypeptides having REFERENCE TO ASEQUENCE LISTING acetylxylan esterase activity and polynucleotides encoding the polypeptides. This application contains a Sequence Listing in computer 15 readable form. The computer readable form is incorporated SUMMARY OF THE INVENTION herein by reference. The present invention relates to isolated polypeptides hav REFERENCE TO A DEPOSIT OF BIOLOGICAL ing acetylxylan esterase activity selected from the group con MATERIAL sisting of: (a) a polypeptide comprising an amino acid sequence hav This application contains a reference to a deposit of bio ing at least 75% identity to the mature polypeptide of SEQID logical material, which deposit is incorporated herein by ref NO: 2; CCC. (b) a polypeptide encoded by a polynucleotide that hybrid 25 izes under at least medium-high stringency conditions with BACKGROUND OF THE INVENTION (i) the mature polypeptide coding sequence of SEQID NO: 1. (ii) the cDNA sequence contained in the mature polypeptide 1. Field of the Invention coding sequence of SEQ ID NO: 1, or (iii) a full-length The present invention relates to isolated polypeptides hav complementary strand of (i) or (ii); ing acetylxylan esterase activity and isolated polynucleotides 30 (c) a polypeptide encoded by a polynucleotide comprising encoding the polypeptides. The invention also relates to a nucleotide sequence having at least 75% identity to the nucleic acid constructs, vectors, and host cells comprising the mature polypeptide coding sequence of SEQID NO: 1; and polynucleotides as well as methods of producing and using (d) a variant comprising a Substitution, deletion, and/or the polypeptides. insertion of one or more (several) amino acids of the mature 2. Description of the Related Art 35 Plant cell wall polysaccharides constitute generally 90% of polypeptide of SEQID NO: 2. the plant cell wall and can be divided into three groups: The present invention also relates to isolated polynucle cellulose, hemicellulose, and pectin. Cellulose represents the otides encoding polypeptides having acetylxylan esterase major constituent of cell wall polysaccharides. Hemicellulo activity, selected from the group consisting of ses are the second most abundant constituent of plant cell 40 (a) a polynucleotide encoding a polypeptide comprising an walls. The major hemicellulose polymer is xylan. The struc amino acid sequence having at least 75% identity to the ture of xylans found in cell walls of plants can differ signifi mature polypeptide of SEQID NO: 2: cantly depending on their origin, but they always contain a (b) a polynucleotide that hybridizes under at least medium beta-1,4-linked D-xylose backbone. The beta-1,4-linked high stringency conditions with (i) the mature polypeptide D-Xylose backbone can be substituted by various side groups, 45 coding sequence of SEQID NO: 1, (ii) the cDNA sequence Such as L-aribinose, D-galactose, acetyl, feruloyl, p-couma contained in the mature polypeptide coding sequence of SEQ royl, and glucuronic acid residues. ID NO: 1, or (iii) a full-length complementary strand of (i) or The biodegradation of the Xylan backbone depends on two (ii); classes of enzymes: endoxylanases and beta-xylosidases. (c) a polynucleotide comprising a nucleotide sequence Endoxylanases (EC 3.2.1.8) cleave the Xylan backbone into 50 having at least 75% identity to the mature polypeptide coding Smaller oligosaccharides, which can be further degraded to sequence of SEQID NO: 1; and xylose by beta-xylosidases (EC 3.2.1.37). Other enzymes (d) a polynucleotide encoding a variant comprising a Sub involved in the degradation of Xylan include, for example, stitution, deletion, and/or insertion of one or more (several) acetylxylan esterase, arabinase, alpha-glucuronidase, feru amino acids of the mature polypeptide of SEQID NO: 2. loyl esterase, and p-coumaric acid esterase. 55 The present invention also relates to nucleic acid con Acetylxylan esterase (EC 3.1.1.6) removes the O-acetyl structs, recombinant expression vectors, recombinant host groups from positions 2 and/or 3 on the beta-D-Xylopyrano cells comprising the polynucleotides, and methods of produc Syl residues of acetylxylan. Acetylxylan plays an important ing a polypeptide having acetylxylan esterase activity. role in the hydrolysis of Xylan because the acetyl side groups The present invention also relates to methods of inhibiting can interfere sterically with the approach of enzymes that 60 the expression of a polypeptide having acetylxylan esterase cleave the backbone. Removal of the acetyl side groups facili activity in a cell, comprising administering to the cell or tates the action of endoxylanases. A classification system for expressing in the cell a double-stranded RNA (dsRNA) mol carbohydrate esterases, based on sequence similarity, has led ecule, wherein the dsRNA comprises a subsequence of a to the definition of 13 families, seven of which contain polynucleotide of the present invention. The present also acetylxylan esterases (Henrissat B., 1991, Biochem. J. 280: 65 relates to a double-stranded inhibitory RNA (dsRNA) mol 309-316, and Henrissat and Bairoch, 1996, Biochem. J. 316: ecule, wherein optionally the dsRNA is a siRNA or a miRNA 695-696). molecule. US 8,129,590 B2 3 4 The present invention also relates to methods for degrading pure, preferably at least 5% pure, more preferably at least a Xylan-containing material with a polypeptide having 10% pure, more preferably at least 20% pure, more preferably acetylxylan esterase activity.