WO2013166312A1 - Enzymes de production de biocarburants et leurs utilisations - Google Patents

Enzymes de production de biocarburants et leurs utilisations Download PDF

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WO2013166312A1
WO2013166312A1 PCT/US2013/039306 US2013039306W WO2013166312A1 WO 2013166312 A1 WO2013166312 A1 WO 2013166312A1 US 2013039306 W US2013039306 W US 2013039306W WO 2013166312 A1 WO2013166312 A1 WO 2013166312A1
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seq
ascomycota
node
amino acid
acid sequence
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PCT/US2013/039306
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English (en)
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Julio M. Fernandez
Raul Perez-Jimenez
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The Trustees Of Columbia University In The City Of New York
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Publication of WO2013166312A1 publication Critical patent/WO2013166312A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)

Definitions

  • Industrial enzymes have many applications in detergents, textile production, pharmaceuticals, biofuel production, as well as many other applications. Industrial enzymes can be limited by their stability at a wide range of temperatures and pH levels, and currently there is no reliable method to broaden the range of use without simultaneously affecting the enzyme's activity. A common practice involves randomly inserting mutations in existing enzymes and screening for variants that exhibit the desired characteristics; however, due to the enormous combinatorial possibilities, this can become costly and work-intensive and does not guarantee success.
  • the invention relates to a modified method to predictably alter and optimize enzymes, mainly by identifying and resurrecting suitable ancestral strains. By examining the molecular and evolutionary biology involved in the enzyme chemistry, this method establishes a fast and economically efficient system to develop and/or improve enzymes, especially industrial enzymes used in harsh environments such as high temperature or low pH.
  • the ancestral cellulases can be classified as cellobiohydrolases II. Modern cellobiohydrolases from fungi are used in the biofuel industry to produce cellulosic ethanol.
  • the invention is based, at least in part, on the discovery of the resurrection of four ancestral cellulase enzymes from fungi.
  • These enzymes are useful for the production of Cellulosic Ethanol for biofuels.
  • Some aspects of the present invention provide for ancestral fungal cellulases. Cellulase enzymes are useful for the production of cellulosic ethanol for bio fuels. In some embodiments, ancestral cellulases can be used for the hydrolysis of carbohydrate polymers that comprise cellulose. Some aspects of the present invention provide for microorganisms that express an ancestral cellulase. Microorganisms are useful for the production of cellulosic ethanol for biofuels. In some embodiments, microorganisms can be used for the hydrolysis and/or fermentation of cellulose.
  • the present invention provides for an isolated polypeptide comprising about 90% identity to the amino acid sequence of SEQ ID NO: 2.
  • the present invention provides for an isolated polypeptide comprising about 90% identity to the amino acid sequence of SEQ ID NO: 3.
  • the present invention provides for an isolated polypeptide comprising about 90% identity to the amino acid sequence of SEQ ID NO: 10.
  • the present invention provides for an isolated polypeptide comprising about 90% identity to the amino acid sequence of SEQ ID NO: 11.
  • the present invention provides for an isolated polypeptide comprising about 90%> identity to any one of the amino acid sequences of SEQ ID NO: 1,
  • SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
  • SEQ ID NO: 28 SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 122, SEQ ID NO: 123, SEQ
  • SEQ ID NO: 179 SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ
  • SEQ ID NO: 229 SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, or SEQ ID NO: 233.
  • the signal peptide of the isolated polypeptide is removed.
  • the present invention provides for an isolated polypeptide comprising about 90% identity to the amino acid sequence of SEQ ID NO: 31.
  • the present invention provides for an isolated polypeptide comprising about 90% identity to the amino acid sequence of SEQ ID NO: 32.
  • the present invention provides for an isolated polypeptide comprising about 90%> identity to the amino acid sequence of SEQ ID NO: 39.
  • the present invention provides for an isolated polypeptide comprising at least about 90% identity to the amino acid sequence of SEQ ID NO: 40.
  • the present invention provides for an isolated polypeptide comprising about 90%> identity to any one of the amino acid sequences of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, or SEQ ID NO: 59.
  • the present invention provides for an nucleic acid encoding a polypeptide of the present invention.
  • the present invention provides for a recombinant
  • the present invention provides for a recombinant
  • the recombinant microorganism is a fungus.
  • the recombinant microorganism is from the phylum Basidomycota, from the phylum Ascomycota, from the subkingdom dikarya, or from the class Sordariomycetes.
  • the recombinant microorganism is a yeast.
  • the recombinant microorganism is a bacteria.
  • the recombinant microorganism is Saccharomyces cerevisiae.
  • the recombinant microorganism is selected from the group consisting of Saccharomyces sp., Pichia sp., Sclerotium rolfsii, Phanenerochate chrysosporium,
  • Trichoderma sp. Aspergillus sp., Schizophyllum sp., and Penicillium sp. In some
  • the recombinant microorganism is selected from the group consisting of E.coli sp., Clostridium sp., Cellulomonas sp., Bacillus sp., Thermomonospora sp., Ruminococcus sp., Bacteriodes sp., Erwinia sp., Acetovibrio sp., Microbispora sp., and Streptomyces sp.
  • the present invention provides for a method for the production of cellulosic ethanol, comprising adding an isolated polypeptide of the present invention, or a combination thereof, to a source material of cellulose for cellulose processing.
  • the method further comprises adding a recombinant microorganism of the present invention, or a combination thereof.
  • the isolated polypeptide and recombinant microorganism are added sequentially, in any order.
  • the isolated polypeptide and recombinant microorganism are added simultaneously.
  • carbohydrate polymers are depolymerized.
  • the present invention provides for a method for the production of cellulosic ethanol, comprising adding a recombinant microorganism of the present invention, or a combination thereof, to a source material of cellulose for cellulose processing.
  • the method further comprises adding a polypeptide of the present invention, or a combination thereof.
  • the isolated polypeptide and recombinant microorganism are added sequentially, in any order.
  • the isolated polypeptide and recombinant microorganism are added simultaneously.
  • carbohydrate polymers are depolymerized.
  • the present invention provides for a method for cellulose processing, comprising adding a polypeptide of the present invention, or a combination thereof, to a source material of cellulose.
  • the method further comprises adding a recombinant microorganism of the present invention, or a combination thereof.
  • the isolated polypeptide and recombinant microorganism are added sequentially, in any order.
  • the isolated polypeptide and recombinant microorganism are added simultaneously.
  • carbohydrate polymers are depolymerized.
  • the present invention provides for a method for cellulose processing, comprising adding a recombinant microorganism of the present invention, or a combination thereof, to a source material of cellulose.
  • the method further comprises adding a polypeptide of the present invention, or a combination thereof.
  • the isolated polypeptide and recombinant microorganism are added sequentially, in any order.
  • the isolated polypeptide and recombinant microorganism are added simultaneously.
  • carbohydrate polymers are depolymerized.
  • FIG. 1 is a phylogenetic tree of fungal cellulases.
  • FIG. 2 is a phylogenetic tree of fungal cellulases.
  • FIG. 3 is a phylogenetic tree of fungal cellulases obtained using BEAST.
  • FIG. 4 is a phylogenetic tree of fungal cellulases obtained using MrBayes.
  • Cellulases are enzymes that can catalyze the hydrolysis of the ⁇ -1,4 glucosidic bonds in cellulose, the predominant component of plant matter. In nature, cellulases facilitate microbial conversion of insoluble cellulose contained within biomass into soluble sugars (EA Bayer et al. Current Opinion in Structural Biology, 8:548-557, 1998).
  • Cellobiohydrolases from fungi can be used in the biofuel industry to produce cellulosic ethanol. Before the sugars in lignocellulosic biomass, such as wood, can be fermented into ethanol, the lignin that encapsulates the cellulose and the cellulose's unique structural conformation within can be addressed with either acid or enzyme hydrolysis (PC Badger, In: J. Janick and A. Whipkey (eds.), Trends in new crops and new uses. ASHS Press, Alexandria, VA. 2002.)
  • the sequences in List #3 are new ancestral sequences for the specified nodes. They contain restriction sites at the termini.
  • the DNA sequence gctagc encodes a restriction site for the Nhel enzyme and the sequence ggtacc encodes a restriction site for the restriction enzyme Kpnl.
  • the protein sequences are in capital letters. These sequences are depicted in Appendix 3.
  • the sequences used in the trees and the sequence alignment are shown in List #4, the phylogenetic trees are shown in FIGS.3 and 4, and the resurrected sequences for each tree are shown in List #5 (BEAST tree) and List #6 (MrBayes tree). These sequences present a reconstruction of fungal cellulase enzymes to be used in the production of bioethanol as a green-fuel source.
  • an "ancestral cellulase molecule” refers to an ancestral cellulose protein, or a fragment thereof.
  • An “ancestral cellulase molecule” can also refer to a nucleic acid (including, for example, genomic DNA, complementary DNA (cDNA), synthetic DNA, as well as any form of corresponding RNA) which encodes a polypeptide corresponding to an ancestral cellulase protein, or fragment thereof.
  • an ancestral cellulose molecule comprises the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO:
  • SEQ ID NO: 186 SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO:
  • an ancestral cellulase molecule can be encoded by a recombinant nucleic acid encoding an ancestral cellulase protein, or fragment thereof.
  • the ancestral cellulase molecules of the invention can be obtained from various sources and can be produced according to various techniques known in the art.
  • a nucleic acid that encodes an ancestral cellulase molecule can be obtained by synthetic or semi-synthetic methods, by screening DNA libraries, or by amplification from a natural source.
  • An ancestral cellulase molecule can include a fragment or portion of an ancestral cellulase protein.
  • An ancestral cellulase molecule can include a variant of the above described examples, such as a fragment thereof.
  • an ancestral cellulase molecule comprises a variant of an ancestral cellulase protein or polypeptide encoded by an ancestral cellulase nucleic acid sequence wherein the variant has an amino acid identity to SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,
  • Such variants can include those having at least from about 46% to about 50% identity to SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,
  • protein variants can include amino acid sequence modifications.
  • amino acid sequence modifications fall into one or more of three classes: substitutional, insertional or deletional variants.
  • Insertions can include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence.
  • variants ordinarily are prepared by site-specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.
  • an ancestral cellulase molecule can be modified by deletion of the sequence encoding the signal peptide.
  • Signal peptides are polypeptide sequences variable in length and amino acid composition found at the amino-terminus of some proteins. Signal peptides direct the secretion of polypeptide molecules through a prokaryotic or eukaryotic cell membrane.
  • Signal peptides have a tripartite structure consisting of a hydrophobic core flanked by a positively charged n-region and a neutral but polar c-region on either side (Tuteja, R., (2005) Arch. BioChem. Biophys. 441 : 107-111).
  • Signal peptide sequences can be identified by various methods, known to one of skill in the art. For example, signal peptide sequences within a polypeptide sequence can be identified using various prediction tools including, but not limited to, Phobius (http://phobius.sbc.su.se/), Predotar (http://urgi.versailles.inra.fr/predotar/predotar.html), SignalP
  • an ancestral cellulase molecule comprises a protein or polypeptide encoded by a nucleic acid sequence encoding an ancestral cellulase protein, such as the sequences shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,
  • an ancestral cellulase molecule can be encoded by a recombinant nucleic acid encoding an ancestral cellulase protein, or fragment thereof.
  • the nucleic acid can be any type of nucleic acid, including genomic DNA, complementary DNA (cDNA), synthetic or semi-synthetic DNA, as well as any form of corresponding RNA.
  • a nucleic acid encoding an ancestral cellulase protein can comprise a recombinant nucleic acid encoding such a protein.
  • the nucleic acid can be a non-naturally occurring nucleic acid created artificially (such as by assembling, cutting, ligating or amplifying sequences).
  • Restriction enzymes can be used to cut nucleic acid sequences in a sequence specific manner, as is known in the art. Restriction enzyme recognition sequences can be added to the ends of a nucleic acid sequence encoding an ancestral cellulase protein (e.g. SEQ ID NOS: 60, 61, 62 and 63).
  • the nucleic acid sequence of a restriction enzyme site can encode amino acids. Amino acids encoded by a restriction enzyme site can form part of the sequence of an ancestral cellulase protein, or may encode additional amino acids at the ends of a polypeptide sequence of an ancestral cellulase protein. Nucleic acid sequences can be double-stranded or single-stranded.
  • the invention further provides for nucleic acids that are complementary to an ancestral cellulase molecule.
  • Complementary nucleic acids can hybridize to the nucleic acid sequence described above under stringent hybridization conditions.
  • stringent hybridization conditions include temperatures above 30°C, above 35°C, in excess of 42°C, and/or salinity of less than about 500 mM, or less than 200 mM.
  • Hybridization conditions can be adjusted by the skilled artisan via modifying the temperature, salinity and/or the concentration of other reagents such as SDS or SSC.
  • an ancestral cellulase molecule can be added to a source material of cellulose for cellulose processing.
  • an ancestral cellulase molecule can be added as an isolated recombinant protein.
  • molecule can be added as an isolated modified recombinant protein.
  • an ancestral cellulase protein, or fragment thereof can be modified by removal of the signal peptide.
  • an isolated polypeptide comprising about 90% identity to the amino acid sequence of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167
  • an ancestral cellulase molecule can be added to a source material of cellulose for cellulose processing by addition of a recombinant microorganism that expresses a nucleic acid encoding an ancestral cellulase protein, or fragment thereof.
  • an ancestral cellulase molecule can be added to a source material of cellulose for cellulose processing by addition of a recombinant microorganism that expresses a nucleic acid encoding an amino acid sequence of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149
  • an ancestral cellulase molecule can be added to a source material of cellulose for cellulose processing by addition of a recombinant protein, or by addition of a recombinant microorganism that expresses a nucleic acid encoding an ancestral cellulase protein, or a combination thereof.
  • the recombinant protein and the recombinant microorganism can be added sequentially, in any order, or simultaneously.
  • the invention utilizes conventional molecular biology, microbiology, and recombinant DNA techniques available to one of ordinary skill in the art. Such techniques are well known to the skilled worker and are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover, ed., 1985); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Nucleic Acid Hybridization” (B. D. Hames & S. J. Higgins, eds., 1985); “Transcription and Translation” (B. D. Hames & S. J.
  • an ancestral cellulase e.g., a molecule comprising the amino acid sequence shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 16
  • 229, 230, 231, 232, or 233) in several ways, which include, but are not limited to, isolating the protein via biochemical means or expressing a nucleotide sequence encoding the protein of interest by genetic engineering methods.
  • the invention provides for ancestral cellulase molecules that are encoded by nucleotide sequences.
  • the ancestral cellulase molecule can be a polypeptide encoded by a nucleic acid (including genomic DNA, complementary DNA (cDNA), synthetic DNA, as well as any form of corresponding RNA).
  • an ancestral cellulase molecule can be encoded by a recombinant nucleic acid encoding an ancestral cellulase protein, or fragment thereof.
  • the ancestral cellulase molecules of the invention can be obtained from various sources and can be produced according to various techniques known in the art.
  • the ancestral cellulase molecule of the invention can be produced via recombinant DNA technology and such recombinant nucleic acids can be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof.
  • a nucleic acid that encodes an ancestral cellulase molecule can be obtained by screening DNA libraries, or by amplification from a natural source.
  • a nucleic acid amplified from a natural source is modified by various mutagenesis methods known in the art to obtain the ancestral cellulase molecules of the invention.
  • an ancestral cellulase molecule can be "codon-optimized," as known in the art.
  • An ancestral cellulase molecule can be a fragment of ancestral cellulase protein.
  • the ancestral cellulase protein fragment can encompass any portion of at least about 8 consecutive amino acids of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,
  • the fragment can comprise at least about 10 consecutive amino acids, at least about 20 consecutive amino acids, at least about 30 consecutive amino acids, at least about 40 consecutive amino acids, a least about 50 consecutive amino acids, at least about 60 consecutive amino acids, at least about 70 consecutive amino acids, at least about 80 consecutive amino acids, at least about 90 consecutive amino acids, at least about 100 consecutive amino acids, at least about 110 consecutive amino acids, at least about 120 consecutive amino acids, at least about 130 consecutive amino acids, at least about 140 consecutive amino acids, at least about 150 consecutive amino acids, at least about 200 consecutive amino acids, at least about 250 consecutive amino acids, at least about 300 consecutive amino acids, at least about 350 consecutive amino acids, or at least about 400 consecutive amino acids of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
  • Fragments include all possible amino acid lengths between about 8 and about 400 amino acids, for example, lengths between about 10 and about 400 amino acids, between about 15 and about 400 amino acids, between about 20 and about 400 amino acids, between about 35 and about 400 amino acids, between about 40 and about 400 amino acids, between about 50 and about 400 amino acids, between about 70 and about 400 amino acids, between about 100 and about 400 amino acids, between about 200 and about 400 amino acids, between about 300 and about 400 amino acids, or between about 350 and about 400 amino acids.
  • a fragment of a nucleic acid sequence that comprises an ancestral cellulase molecule can encompass any portion of at least about 8 consecutive nucleotides. In one embodiment, the fragment can comprise at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, or at least about 30 nucleotides.
  • the ancestral cellulase molecules can be recombinant enzymes, and can be produced in a variety of ways known in the art.
  • polypeptides e.g., a molecule comprising the amino acid sequence shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150
  • the nucleic acid is expressed in an expression cassette, for example, to achieve overexpression in a cell.
  • the nucleic acids of the invention can be an R A, cDNA, cDNA-like, or a DNA of interest in an expressible format, such as an expression cassette, which can be expressed from a natural promoter or an entirely
  • the nucleic acid of interest can encode a protein, and may or may not include introns. Any recombinant expression system can be used, including, but not limited to, the recombinant microorganisms of the invention, as well as other bacterial, fungal, mammalian, insect, or plant cell expression systems.
  • Nucleic acid sequences comprising an ancestral cellulase molecule that encode a polypeptide can be synthesized, in whole or in part, using chemical methods known in the art.
  • an ancestral cellulase molecule can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques. Protein synthesis can either be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).
  • fragments of an ancestral cellulase molecule can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • Host cells transformed with a nucleic acid sequence encoding an ancestral cellulase molecule such as, e.g., a molecule comprising the amino acid sequence shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,
  • the polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • Methods for protein production by recombinant technology in different host systems are well known in the art (Sambrook, et al., "Molecular Cloning: a Laboratory Manual” (2001); Gellissen, G., “Novel Microbial and Eukaryotic Expression Systems” (2005)).
  • Expression vectors containing a nucleic acid sequence encoding an ancestral cellulase molecule can be designed to contain signal sequences which direct secretion of soluble polypeptide molecules encoded by an ancestral cellulase molecule, through a prokaryotic or eukaryotic cell membrane.
  • An ancestral cellulase molecule can be produced as an extracellular enzyme that is secreted into the culture medium, from which it can easily be recovered and isolated.
  • the spent culture medium of the production host can be used as such, or the host cells can be removed therefrom, and/or it can be concentrated, filtrated or fractionated. It can also be dried.
  • an ancestral cellulase molecule, or fragment thereof can be modified by removal of the signal peptide which can allow the polypeptide molecules to be contained intracellularly.
  • An isolated polypeptide of the present invention includes, but is not limited to, culture medium containing the polypeptide from which cells and cell debris have been removed.
  • the polypeptides can be isolated e.g. by adding anionic and/or cationic polymers to the spent culture medium to enhance precipitation of cells, cell debris and other unwanted enzymes.
  • the medium can be filtrated using an inorganic filtering agent and a filter to remove the precipitants formed.
  • the filtrate can be further processed using a semi-permeable membrane to remove excess of salts, sugars and metabolic products.
  • a synthetic peptide can be substantially purified via high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the composition of a synthetic ancestral cellulase molecule can be confirmed by amino acid analysis or sequencing. Additionally, any portion of an amino acid sequence comprising a protein encoded by an ancestral cellulase molecule can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.
  • the invention further encompasses methods for using a protein or polypeptide encoded by a nucleic acid sequence of an ancestral cellulase molecule.
  • the polypeptide can be modified, such as by glycosylations and/or acetylations and/or chemical reaction or coupling, and can contain one or several non-natural or synthetic amino acids.
  • An example of an ancestral cellulase molecule comprises the amino acid sequence shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,
  • Some aspects of the present invention provide for recombinant microorganisms that express a nucleic acid encoding an ancestral cellulase enzyme (e.g., the amino acid sequence shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,
  • microorganisms can include both prokaryotic and eukaryotic microorganisms, such as bacteria and yeast.
  • the microorganism is a fungus.
  • the microorganism is from the phylum Basidomycota, from the phylum
  • the microorganism is a yeast. In yet another embodiment, the microorganism is a bacteria. In another embodiment, the microorganism is E.Coli sp., Clostridium sp., Cellulomonas sp., Bacillus sp., Thermomonospora sp., Ruminococcus sp., Bacteriodes sp., Erwinia sp., Acetovibrio sp., Microbispora sp., Streptomyces sp.
  • the microorganism is Saccharomyces sp., Pichia sp., Sclerotium rolfsii, Phanenerochate chrysosporium, Trichoderma sp., Aspergillus sp., Schizophyllum sp., and Penicillium sp.
  • a microorganism is a eukaryotic or prokaryotic microorganism.
  • a microorganism is a yeast, such as Saccharomyces cerevisiae.
  • a microorganism is a bacteria, such as a gram-positive bacteria or a gram-negative bacteria.
  • microorganisms may be used according to the present invention.
  • other organisms from the genera Achaetomium, Acremonium, Aspergillus,
  • Botrytis Chaetomium, Chrysosporium, Collybia, Fames, Fusarium, Humicola, Hypocrea, Lentinus, Metanacarpus, Myceliophthora, Myriococcum, Neurospora, Penicillium,
  • Phanerochaete Phlebia, Pleurotus, Podospora, Polyporus, Pycnoporus, Rhizoctonia,
  • Additional organisms include, but are not limited to Acetobacter aceti, Achromobacter, Acidiphilium,
  • chrysanthemi Gliconobacter, Gluconacetobacter, Haloarcula, Humicola insolens, Humicola nsolens, Kitasatospora setae, Klebsiella, Klebsiella oxytoca, Kluyveromyces, Kluyveromyces fragilis, Kluyveromyces lactis, Kocuria, Lactlactis, Lactobacillus, Lactobacillus fermentum, Lactobacillus sake, Lactococcus, Lactococcus lactis, Leuconostoc, Methylocystis,
  • Microbacterium imperiale, Micrococcus lysodeikticus, Microlunatus, Mucor javanicus, Mycobacterium, Myrothecium, Nitrobacter, Nitrosomonas, Nocardia, Papaya carica,
  • Pediococcus Pediococcus halophilus, Penicillium, Penicillium camemberti, Penicillium citrinum, Penicillium emersonii, Penicillium roqueforti, Penicillum lilactinum, Penicillum multicolor, Paracoccus pantotrophus, Propionibacterium, Pseudomonas, Pseudomonas fluorescens, Pseudomonas denitrificans, Pyrococcus, Pyrococcus furiosus, Pyrococcus horikoshii, Rhizobium, Rhizomucor miehei, Rhizomucor pusillus Lindt, Rhizopus, Rhizopus delemar, Rhizopus japonicus, Rhizopus niveus, Rhizopus oryzae, Rhizopus oligosporus, Rhodococcus, Sccharomyces cerevisiae, Sclerotina libertina
  • a recombinant microorganism may be engineered to secrete an ancestral cellulase molecule into the culture media, such as by incorporating a signal peptide or an autotransporter domain into the ancestral cellulase molecule.
  • ancestral cellulase molecules can be fused with any combination of signal peptides and or autotransporter domains found in secreted proteins as is known in the art.
  • ancestral cellulase molecules can be designed to maximize the secretion of ancestal cellulase molecules into the culture media, and may also include the use of many different linker sequences that fuse signal peptides, ancestal cellulase molecules, and autotransporters that improve the efficiency of secretion or the cell surface presentation.
  • an ancestral cellulase molecule can be modified by deletion of the sequence encoding the signal peptide.
  • an ancestral cellulase molecule is purified from the culture media. In other embodiments, an ancestral cellulase molecule is not purified from the culture media.
  • any other recombinant expression system can be used to obtain an isolated ancestral cellulase molecule.
  • Bacterial Expression Systems One skilled in the art understands that expression of desired protein products in prokaryotes is most often carried out in E. coli with vectors that contain constitutive or inducible promoters.
  • Some non-limiting examples of bacterial cells for transformation include E.Coli sp., Clostridium sp., Cellulomonas sp., Bacillus sp., Thermomonospora sp., Ruminococcus sp., Bacteriodes sp., Erwinia sp., Acetovibrio sp., Microbispora sp., Streptomyces sp., and the bacterial cell line E.
  • E. coli strains DH5 or MC1061/p3 (Invitrogen Corp., San Diego, Calif), which can be transformed using standard procedures practiced in the art, and colonies can then be screened for the appropriate plasmid expression.
  • a number of expression vectors can be selected.
  • Non- limiting examples of such vectors include multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene).
  • Some E. coli expression vectors (also known in the art as fusion- vectors) are designed to add a number of amino acid residues, usually to the N-terminus of the expressed recombinant protein.
  • Such fusion vectors can serve three functions: 1) to increase the solubility of the desired recombinant protein; 2) to increase expression of the recombinant protein of interest; and 3) to aid in recombinant protein purification by acting as a ligand in affinity purification.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified, may also be used.
  • fusion expression vectors include pGEX, which fuse glutathione S-tranf erase (GST) to desired protein; pcDNA 3.1/V5-His A B & C (Invitrogen Corp, Carlsbad, CA) which fuse 6x-His to the recombinant proteins of interest; pMAL (New England Biolabs, MA) which fuse maltose E binding protein to the target recombinant protein; the E.
  • coli expression vector pUR278 (Ruther et al., (1983) EMBO 12: 1791), wherein the coding sequence may be ligated individually into the vector in frame with the lac Z coding region in order to generate a fusion protein; and pIN vectors (Inouye et al, (1985) Nucleic Acids Res. 13:3101-3109; Van Heeke et al, (1989) J. Biol. Chem.
  • Fusion proteins generated by the likes of the above-mentioned vectors are generally soluble and can be purified easily from lysed cells via adsorption and binding of the fusion protein to an affinity matrix.
  • fusion proteins can be purified from lysed cells via adsorption and binding to a matrix of glutathione agarose beads subsequently followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target can be released from the GST moiety.
  • an ancestral cellulase molecule is not purified from the culture media.
  • Plant and Insect Expression Systems Other suitable cell lines, in addition to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences for an ancestral cellulase molecule may alternatively be used to produce the molecule of interest.
  • a non-limiting example includes plant cell systems infected with recombinant virus expression vectors (for example, tobacco mosaic virus, TMV; cauliflower mosaic virus, CaMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing coding sequences for an ancestral cellulase molecule.
  • sequences encoding an ancestral cellulase molecule can be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from tobacco mosaic virus TMV.
  • plant promoters such as the small subunit of RUBISCO or heat shock promoters, can be used. These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection.
  • an insect system also can be used to express an ancestral cellulase molecule.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • Sequences encoding a trefoil family molecule can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
  • Successful insertion of the nucleic acid sequences of an ancestral cellulase molecule will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which an ancestral cellulase molecule can be expressed.
  • a fungal system also can be used to express an ancestral cellulase molecule.
  • Fungi can be transformed with recombinant fungal expression vectors containing coding sequences for an ancestral cellulase molecule.
  • Some non- limiting examples of fungi for transformation include, Saccharomyces sp., Pichia sp., Sclerotium rolfsii, Phanenerochate chrysosporium, Trichoderma sp., Aspergillus sp., Schizophyllum sp., and Penicillium sp..
  • fungi from the subkingdom dikarya, from the phylum Basidomycota, from the phylum Ascomycota, or from the class Sordariomycetes can be transformed with recombinant fungal expression vectors containing coding sequences for an ancestral cellulase molecule.
  • Mammalian Expression Systems Mammalian cells can also contain an expression vector (for example, one that harbors a nucleotide sequence encoding an ancestral cellulase molecule for expression of a desired product.
  • Expression vectors containing such a nucleic acid sequence linked to at least one regulatory sequence in a manner that allows expression of the nucleotide sequence in a host cell can be introduced via methods known in the art.
  • the vector can be a recombinant DNA or RNA vector, and includes DNA plasmids or viral vectors.
  • a number of viral-based expression systems can be used to express an ancestral cellulase molecule in mammalian host cells (e.g., adeno-associated virus, retrovirus, adenovirus, lentivirus or alphavirus).
  • mammalian host cells e.g., adeno-associated virus, retrovirus, adenovirus, lentivirus or alphavirus.
  • Regulatory sequences are well known in the art, and can be selected to direct the expression of a protein or polypeptide of interest (such as an ancestral cellulase molecule) in an appropriate host cell as described in Goeddel, Gene Expression Technology: Methods in Enzvmology 185, Academic Press, San Diego, Calif. (1990).
  • Non- limiting examples of regulatory sequences include: polyadenylation signals, promoters, enhancers, and other expression control elements. Practitioners in the art understand that designing an expression vector can depend on factors, such as the choice of host cell to be transfected and/or the type and/
  • Enhancer regions which are those sequences found upstream or downstream of the promoter region in non-coding DNA regions, are also known in the art to be important in optimizing expression. If needed, origins of replication from viral sources can be employed, such as if a prokaryotic host is utilized for introduction of plasmid DNA. However, in eukaryotic organisms, chromosome integration is a common mechanism for DNA replication.
  • a gene that encodes a selectable marker (for example, resistance to antibiotics or drugs, such as ampicillin, neomycin, G418, and hygromycin) can be introduced into host cells along with the gene of interest in order to identify and select clones that stably express a gene encoding a protein of interest.
  • the gene encoding a selectable marker can be introduced into a host cell on the same plasmid as the gene of interest or can be introduced on a separate plasmid. Cells containing the gene of interest can be identified by drug selection wherein cells that have incorporated the selectable marker gene will survive in the presence of the drug. Cells that have not incorporated the gene for the selectable marker die. Surviving cells can then be screened for the production of the desired protein molecule (for example, an ancestral cellulase molecule).
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed ancestral cellulase molecule in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities can be chosen to ensure the correct modification and processing of the foreign protein.
  • An exogenous nucleic acid can be introduced into a cell via a variety of techniques known in the art, such as lipofection, microinjection, calcium phosphate or calcium chloride precipitation, DEAE-dextrin-mediated transfection, or electroporation. Electroporation is carried out at approximate voltage and capacitance to result in entry of the DNA construct(s) into cells of interest. Other methods used to transfect cells can also include modified calcium phosphate precipitation, polybrene precipitation, liposome fusion, and receptor-mediated gene delivery.
  • a host cell strain which modulates the expression of the inserted sequences, or modifies and processes the nucleic acid in a specific fashion desired also may be chosen. Such modifications (for example, glycosylation and other post-translational modifications) and processing (for example, cleavage) of protein products may be important for the function of the protein.
  • Different host cell strains have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. As such, appropriate host systems or cell lines can be chosen to ensure the correct modification and processing of the foreign protein expressed, such as an ancestral cellulase molecule.
  • eukaryotic host cells possessing the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • Non-limiting examples of host cells include E.Coli sp., Clostridium sp., Cellulomonas sp., Bacillus sp., Thermomonospora sp., Ruminococcus sp., Bacteriodes sp., Erwinia sp., Acetovibrio sp., Microbispora sp., Streptomyces sp., Saccharomyces sp., Pichia sp., Sclerotium rolfsii, Phanenerochate chrysosporium, Trichoderma sp., Aspergillus sp., Schizophyllum sp., and Penicillium sp..
  • Various culturing parameters can be used with respect to the host cell being cultured. Appropriate culture conditions for host cells are well known in the art or can be determined by the skilled artisan (see, for example, Madigan M. et al., "Brock Biology of Microorganisms", 2012). Cell culturing conditions can vary according to the type of host cell selected. Commercially available medium can be utilized.
  • Cells suitable for culturing can contain introduced expression vectors, such as plasmids or viruses.
  • the expression vector constructs can be introduced via transformation, microinjection, trans fection, lipofection, electroporation, or infection.
  • the expression vectors can contain coding sequences, or portions thereof, encoding the proteins for expression and production.
  • Expression vectors containing sequences encoding the produced proteins and polypeptides, as well as the appropriate transcriptional and translational control elements, can be generated using methods well known to and practiced by those skilled in the art. These methods include synthetic techniques, in vitro recombinant DNA techniques, and in vivo genetic recombination which are described in J.
  • An ancestral cellulase molecule (such as, e.g., a molecule comprising the amino acid sequence shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,
  • a purified ancestral cellulase molecule can be separated from other compounds which normally associate with the ancestral cellulase molecules, in the cell, such as certain proteins, carbohydrates, or lipids, using methods practiced in the art.
  • the cell culture medium or cell lysate is centrifuged to remove particulate cells and cell debris.
  • the desired polypeptide molecule (for example, an ancestral cellulase molecule) is isolated or purified away from contaminating soluble proteins and polypeptides by suitable purification techniques.
  • Non-limiting purification methods for proteins include: size exclusion chromatography; affinity chromatography; ion exchange chromatography; ethanol precipitation; reverse phase HPLC; chromatography on a resin, such as silica, or cation exchange resin, e.g., DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, e.g., Sephadex G-75, Sepharose; and the like.
  • Other additives such as protease inhibitors (e.g., PMSF or proteinase K) can be used to inhibit proteolytic degradation during purification.
  • Purification procedures that can select for carbohydrates can also be used, e.g., ion-exchange soft gel chromatography, or HPLC using cation- or anion-exchange resins, in which the more acidic fraction(s) is/are collected.
  • ion-exchange soft gel chromatography or HPLC using cation- or anion-exchange resins, in which the more acidic fraction(s) is/are collected.
  • HPLC cation- or anion-exchange resins
  • Some aspects of the present invention provide for ancestral fungal cellulases.
  • Cellulase enzymes are useful for the production of cellulosic ethanol for biofuels.
  • ancestral cellulases can be used for the hydrolysis of carbohydrate polymers that comprise cellulose.
  • microorganisms that express an ancestral cellulase. Microorganisms are useful for the production of cellulosic ethanol for biofuels. In some embodiments, microorganisms can be used for the hydrolysis and/or fermentation of cellulose.
  • the production of cellulosic ethanol biofuels from cellulosic materials can be performed by various techniques known in the art. See, for example, Canilha L. et al., (2012) J. Biomed. Biotech., 2012:989572; U.S. Patent No. 8,318,473; U.S. Patent No. 8,409,836; U.S. Patent Application Publication No. 20110171710A1 , which are incorporated by reference in their entireties.
  • the starting material for the production of cellulosic biofuels can be cellulosic materials (i.e. any material comprising lignocellulose, cellulose, hemicellulose, or a combination thereof).
  • a source material of cellulose can be any cellulosic material.
  • cellulosic materials include, but are not limited to, fruits, plants, vegetables, woods, grasses, inedible parts of plants, byproducts of lawn and tree maintenance, corn stover, Panicum virgatum, Miscanthus grass species, wood chips, sugarcane residues, sugarcane bagasse, straw, pulp and paper residues, waste paper, textile fibers (e.g., cotton, linen, hemp, jute) and cellulosic fibers (e.g., modal, viscose, lyocel).
  • textile fibers e.g., cotton, linen, hemp, jute
  • cellulosic fibers e.g., modal, viscose, lyocel
  • cellulosic ethanol During the production of cellulosic ethanol from cellulosic material, cellulosic materials can be processed by various techniques known in the art. Most of the
  • carbohydrates in cellulosic material are in the form of lignocellulose, which can comprise cellulose, hemicellulose, pectin and/or lignin.
  • Cellulosic material can be pre-treated by physical and/or chemical means. Pre-treatment can make the cellulose fraction more accessible to hydrolysis.
  • Cellulose and/or hemicellulose comprising the cellulosic materials can then be hydrolysed into sugars (e.g., glucose).
  • ancestral cellulases can be used for the hydrolysis of cellulose.
  • the carbohydrate polymers of cellulose are depolymerized by an ancestral cellulase.
  • Sugars made available by hydrolysis can be used by microorganisms to produce ethanol by fermentation.
  • the present invention provides a method for the production of cellulosic ethanol from a source material of cellulose.
  • Cellulase enzymes can be added to cellulosic materials by various techniques.
  • ancestral cellulases are added to cellulosic materials as an isolated polypeptide.
  • recombinant microorganisms that express ancestral cellulases are added to cellulosic materials.
  • microorganisms that do not express cellulase enzymes can be genetically modified to express ancestral cellulases.
  • Microorganisms can be modified in a variety of ways, such as, but not limited to, to express cellulases, to express large volumes of cellulases, to express modified cellulases, and to express ancestral cellulases.
  • ancestral cellulases can be added to cellulosic materials by addition of isolated polypeptides and by addition of recombinant microorganisms that express ancestral cellulases. Isolated polypeptides and recombinant microorganisms can be added simultaneously or sequentially, in any order.
  • Ancestral cellulases needed for the hydrolysis of the cellulosic material according to the invention may be added in an enzymatically effective amount either simultaneously e.g. in the form of an enzyme mixture, or sequentially, or in combination with any microorganism of the present invention, or in combination with microorganisms that mediate fermentation.
  • any combination of the ancestral cellulase molecules comprising an amino acid sequence having about 90% identity to SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166
  • the tree is shown in FIG. 1.
  • the list of ancestral sequences are listed as SED ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, and 63.
  • Bayesian analysis is a method used to analyze data that uses previous information in the generation of a functional result. This method of analysis was used in tandem with phylogenic studies in this technology.
  • the sequences used in the trees and the sequence alignment are listed as SED ID NOS: 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, and 121.
  • the phylogenetic trees are shown in FIGS. 3 and 4, and the resurrected sequences for each tree are listed as SEQ ID NOS: 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, and 177, (BEAST tree) and SEQ ID NOS: 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,
  • AAALPVEERQ ACASVWGQCG GQGWSGPTCC ASGSTCWQN PYYSQCLPGS ATTTTTSSST TSRSSSTTST SSTTTTSPPT TTPSTASYSG NPFAGVQLWA NAYYASEVHT LAIPSLTDGA LAAKASAVAK VPSFQWLDTA AKVPTLMAGT LADIRAANKA GANPPYAGQF VVYDLPDRDC AAAASNGEFS IADNGVANYK AYIDAIRAQL VEYSDIRIIL VIEPDSLANM VTNMNVAKCA NAQSAYLECT NYALKQLNLP HVAMYLDAGH AGWLGWPANL QPAATLFAKV YKDAGKPAAV RGLATNVANY NAWSIASPPS YTQGDPNYDE KHYINALAPL LSANGWPDAH FIVDQGRSGK QPTGQQEWGD WCNVIGTGFG VRPTTNTGSS LEDAFV
  • AAALPVEERQ ACASVWGQCG GQGWSGPTCC ASGSTCVVSN PYYSQCLPGS ATPTTTSSST TSRSSSTTSR SSTTTTSPPT TTPSTASYSG NPFAGVNLWA NAYYASEVHT LAIPSLTDGA LAAKASAVAK VPSFQWLDTA AKVPTLMAGT LADIRAANKA GGNPPYAGQF VVYDLPDRDC AAAASNGEFS IADGGVAKYK AYIDAIRAQL VEYSDIRIIL VIEPDSLANM VTNMGVPKCA NAQSAYLECT NYAVKQLNLP HVAMYLDAGH AGWLGWPANL QPAATLFAKV YKDAGKPAAL RGLATNVANY NAWNITSPPS YTQGNPNYDE KHYINALAPL LSANGWPDAH FIVDQGRSGK QPTGQQEWGD WCNVIGTGFG VRPTANTGSS LVDAFV
  • FRMLRYLSIV AATAILTGVE AQQSVWGQCG GQGWSGATSC AAGSTCSTQN PYYAQCIPGT ATSTTSSTTT SSTSASTTTT TTTTTTTAST TTTTTAAASG NPFSGYQLYA NPYYSSEVHT LAIPSLTDGS LAAAATKAAE IPSFVWLDTA AKVPTLMGTY LANIEAANKA GASPPIAGIF VVYDLPDRDC AAAASNGEYT VANNGVANYK AYIDSIVAQL KAYPDVHTIL IIEPDSLANM VTNLSTAKCA EAQSAYYECV NYALINLNLP NVAMYIDAGH AGWLGWSANL SPAAQLFATV YKNASSPAAL RGLATNVANY NAWSISSPPS YTSGDSNYDE KLYINALSPL LTSNGWPNAH FIMDTSRNGV QPTKQQAWGD WCNVIGTGFG VQPTTNTGDP LEDAFV
  • AAQAPVWGQC GGTGWTGPTC CASGSTCWQ NPYYSQCLPG STTSSTTTTS TTTSSPTSST TTTTSTTTPP TTSPTTTTPP PGSTSTPASG NPFAGYQLYL SPYYAAEVAA LAAPNITDPA LKAKAASVAN IPTFTWFDTV AKVPDLGTYL ADASALQKSS GQKPYAVQIV VYDLPDRDCA AAASNGEFSI ANNGMANYKT YIDSIAAQLK KYSDVRWAV IEPDSLANMV TNLNVAKCAN AQTAYKEGVT YALKQLNLVG VYMYLDAGHA GWLGWPANLS PAAQLFAQLY KNAGSPSFVR GLATNVANYN ALSAASPDSY TQGNPNYDEI HYINALAPML SSQGFPPAHF IVDQGRSGVQ NIRQQQWGDW CNVKGAGFGT RPTTNTGSSL IDAIVWVKPG GESDGTSDTS
  • AAQAPVWGQC GGTGWTGPTT CASGSTCWQ NPYYSQCLPG STTSSTTTTS TTTSSPTSST TTTTSTTTPP TGSPTTTTPP PGSTSTPAAG NPFVGYQLYL SPYYAAEVAA LAASNITDPT LKAKAASVAN IPTFTWFDVV AKVPDLGTYL ADASALQKSS GQKPYLVQIV VYDLPDRDCA AAASNGEFSI ANNGMANYKT YIDQIAAQIK KYPDVRVVAV IEPDSLANMV TNLNVAKCAN AQTAYKEGVT YALKQLNSVG VYMYLDAGHA GWLGWPANLS PAAQLFAQLY KNAGSPSFVR GLATNVANYN ALSAASPDPI TQGNPNYDEI HYINALAPML SSQGFPPAHF IVDQGRSGVQ NIRQQQWGDW CNVKGAGFGT RPTTNTGSSL IDAIVWVKPG GES
  • QAQASVWGQC GGQGWSGPTC CASGSTCWQ NPYYSQCLPG STTTSTTTTS TTTTSSTSST TTTTSTTTPP TTSPTTTTPP
  • AASTTASASG NPFSGYQLYA NPYYASEVHS LAIPSLTDGA LAAKASAVAK VPSFVWLDTA AKVPTMGTYL ADIRAANKAG ANPPYAGQFV VYDLPDRDCA AAASNGEFSI ANNGVANYKA YIDSIRAQLV KYSDVRIILV IEPDSLANMV TNLNVAKCAN AQSAYLECVN YALKQLNLPN VAMYLDAGHA GWLGWPANLS PAAQLFAKVY KNAGSPAAVR GLATNVANYN AWSIASPPSY TQGDPNYDEK HYINALAPLL SSNGFPDAHF IVDTGRNGVQ PTRQQEWGDW CNVIGTGFGV RPTTNTGSSL EDAFVWVKPG GESDGTSD
  • QACASQWGQC GGQGWSGPTC CASGSTCWQ NPYYSQCLPG STTTSTTTRS STTTSSVSST TTSTSTTTPP TGSPTTTTPP AASGTASYSG NPFAGVQLWA NAYYASEVHS LAIPSLTDGA LAAKASAVAK VPSFQWLDTA AKVPLMAGTL ADIRAANKAG ANPPYAGQFV VYDLPDRDCA AAASNGEFSI ADNGVANYKA YIDAIRAQLV EYSDIRIILV IEPDSLANMV TNMNVAKCAN AQSAYLECTN YAVKQLNLPN VAMYLDAGHA GWLGWPANLQ PAATLFAKVY KNAGKPAALR GLATNVANYN AWSIASPPSY TQGDPNYDEK HYIQALAPLL SSNGWPDAHF IVDQGRSGKQ PTGQQEWGDW CNVIGTGFGV RPTTNTGSSL EDAFVWVKPG GECDGTSDTT
  • QACASQWGQC GGQGWSGPTC CASGSTCVVS NPYYSQCLPG SATTSSSSTA SSTTSSVRST TTSTSTTTPP TGSPTTTTPP APSGGATYTG NPFAGVNLWA NAYYASEVSS LAIPSLSDGA LATAAAKVAK VPTFQWMDTA AKVPLMDGTL ADIRRANKAG GNPPYAGQFV VYNLPDRDCA AAASNGELSI ADGGVAKYKA YIDAIRAMLV KYSDIRIILV IEPDSLANMV TNMGVPKCAN AQAAYLECTN YAVTQLNLPN VAMYLDAGHA GWLGWPANLQ PAATLFAKVY KDAGKPKALR GLATNVSNYN AWNITSPPSY TQGNPNYDEK HYIEALAPLL SSNGWPDAKF IVDQGRSGKQ PTGQQEWGDW CNAIGTGFGV RPTANTGSSL VDAFVWVKPG GESDGTSDTT AARY
  • SEQ ID NO: 62 (AsCA node #41) gctagcQAQASVWGQC GGQGWSGPTC CASGSTCVVQ NPYYSQCLPG STTTSTTTTS TTTTSSTSST TTTTSTTTPP TTSPTTTTPP AASTTASASG NPFSGYQLYA NPYYASEVHS LAIPSLTDGA LAAKASAVAK VPSFVWLDTA AKVPTMGTYL ADIRAANKAG ANPPYAGQFV VYDLPDRDCA AAASNGEFSI ANNGVANYKA YIDSIRAQLV KYSDVRIILV IEPDSLANMV TNLNVAKCAN AQSAYLECVN YALKQLNLPN VAMYLDAGHA GWLGWPANLS PAAQLFAKVY KNAGSPAAVR GLATNVANYN AWSIASPPSY TQGDPNYDEK HYINALAPLL SSNGFPDAHF IVDTGRNGVQ PTRQQEWGDW CNVIGTGFGV
  • SEQ ID NO: 64 (tr_A9FHT2_Bacteria_Sorangium_cellulosum) AC GDGG-GGDTS GTGGGSSGVG APTSSGVGAG TPTSSGNVDP TTTSSGNVDP
  • NQPTGQSQWG DWCNVKNTGF GVRPTTDTGD ELVDAFVWVK PGGESDGTSD TSAERYDAHC
  • GLA-DALKPA PEAGQWFQAY FEQLLTNANP PF SEQ ID NO: 71 (tr_B2ABX7_Ascomycota_Podospora_anserina)
  • SEQ ID NO: 80 sp_Q9ClS9_Ascomycota_ Humicola insolens
  • VQPTAQQAWG DWCNLIGTGF GVRPTTNTGD ALEDAFVWIK PGGEGDGTSD TTAARYDFHC
  • SEQ ID NO: 90 (tr_G7XQ80 Ascomycota Aspergillus kawachii) QTLWGQC GGQGYSGATS CVAGATCSTI NEYYAQCTPA T-SATTLKTT TSTTTA AMTTT TATSSPAASA SP TTTAS ASGPFSGYQL YVNPYYSSEV ASLAIPSLT-
  • KQPTGQQAWG DWCNVINTGF GVRPTTSTGD DLVDAFVWVK PGGESDGTSD SSATRYDAHC
  • KQPTGQQAWG DWCNVINTGF GERPTTDTGD ALVDAFVWVK PGGESDGTSD SSATRYDAHC
  • GYS-DALQPA PEAGTWFQAY FVQLLTNANP AF SEQ ID NO: 92 (sp_P46236_Ascomycota_Fusarium_oxysporum)
  • SEQ ID NO: 93 sp QOCFPl Ascomycota Aspergillus terreus
  • QTLWGQC GGIGWTGPTN
  • CVAGAACSTQ NPYYAQCLPG TSTTLTTTTR VTTTTTSTTS
  • GDMVAKASAV AKVPSFQWLD TAAKVPTMAD TLADIAKANQ
  • AGASPAYAGL FWYDLPDRD CAAAASNGEY SIADNGVANY KAYI
  • DAIKAQ LVANSDTRIL LVVEPDSLAN LVTNMNVAKC ANAHDAYLEC INYAVTQLNL PNVAMYLDAG HAGWLGWSAN LQPAATLFAN VYSNAGKPAS LRGLATNVAN YNAWTIASAP SYTQGDSNYD
  • EKLYVQALSP LLSSAGW-DA HFITDQSRSG KQPTGQNAWG DWCNVIGTGF GTRPTTDTGL DIEDALVWVK PGGECDGTSN TTAARYDYHC GLS-DALQPS PEAGTWFQAY FVQLLTNANP AF
  • VQPTKQQAWG DWCNVIGTGF GTPFTTDTGD ALQDAFIWVK PGGECDGTSD TSSPRYDAHC
  • VQPTKQQAWG DWCNVIGTGF GVQPTTNTGD PLEDAFVWVK PGGESDGTSN SSATRYDFHC
  • VQPTKQQAWG DWCNVIGTGF GVQPTTNTGD PLEDAFVWVK PGGESDGTSN SSATRYDFHC
  • GYS-GALQPA PEAGTWFQAY FVQLLTNANP AL SEQ ID NO: 99 (sp_Q4WFK4_Ascomycota_Neosartorya_fumigata) QTVWGQC GGQGWSGPTS CVAGAACSTL NPYYAQCIPG A—STTLTTT TAATTT SQTTT KPTTTGPTTS AP TVT ASGPFSGYQL YANPYYSSEV HTLAMPSLP-
  • SSLQPKASAV AEVPSFVWLD VAAKVPTMGT YLADIQAKNK AGANPPIAGI FWYDLPDRD
  • SEQ ID NO: 100 (sp_Q5B2E8_Ascomycota_Emericella_nidulans) QTLYGQC GGSGWTGATS CVAGAACSTL NQWYAQCLPA —ATTTSTTL TTTTSS VTTTS NPGSTTTT— —SSVTVTAT ASGPFSGYQL YVNPYYSSEV QSIAIPSLT-
  • TSVGTTSPAT TPTTKPST TAA ASGPFSGYQL YANPYYSSEV HTLALPSLT-
  • VQPTKQQAWG DWCNVIGTGF GVPPTTNTGD PLEDAFVWVK PGGESDGTSN SSATRYDYHC
  • SEQ ID NO: 102 (sp_AlDJQ7_Ascomycota_Neosartorya_fischeri) QTVWGQC GGQGWSGPTN CVAGAACSTL NPYYAQCIPG ATATSTT LSTTTT TQTTT KPTTTGPTTS AP TVT ASGPFSGYQL YANPYYSSEV HTLAMPSLP-
  • SSLQPKASAV AEVPSFVWLD VAAKVPTMGT YLADIQAKNK AGASPPIAGI FWYDLPDRD
  • CAALASNGEY S IANNGVANY KAYI DAIRAQ LVKYSDVHTI LVIEPDSLAN LVTNLNVAKC ANAQSAYLEC VDYALKQLNL PNVAMYLDAG HAGWLGWPAN LGPAATLFAK VYTDAGSPAA LRGLATNVAN YNAWSLSTCP SYTQGDPNCD EKKYINAMAP LLKNAGF-DA HFIMDTSRNG VQPTKQSAWG DWCNVIGTGF GVRPSTNTGD PLQDAFVWIK PGGESDGTSN SSSARYDAHC GYS-DALQPA PEAGTWFQAY FEQLLTNANP SF
  • QACASQWGQC GGQGWTGPSC CAAGSVCTVS NPFYSQCLPG STVASSTSTV RTSSTPVVSP SRTSTVTGSV STTSAGTGTT PP—PTGGAT YTGPFVGVNL WANSYYASEI STLAIPSLS- PALATAAAKV AKVPTFMWMD TRSKIPLVDA TLADIRKANQ AGAN—YAGE FWYNLPDRD CAAAASNGEL SIADGGVAKY KQYI DDIRAM VVKYSDIRII LTIEPDSLAN LVTNLNVPKC AGAQAAYLEG TNYAVTQLNL PNVAMYLDGG HAGWLGWPAN LPPAAAMYAK VYKDAGKPKA LRGLVTNVSN YNGYSISTAP SYTQGNANYD EKHYIEALAP LLSAEGW-DA KFIVDQGRSG KQPTGQLAWG DWCNAIGTGF GVRPTANTGS TLVDAFVWVK PGGESDG
  • GSA-SSMKP- SEQ ID NO: 106 (gi_345565889_Ascomycota_Arthrobotrys_oligospora) ⁇ LWGQC
  • GGIGWTGATN CVAGAACSTL NPYYAQCLSA AATTPRTTTT PATTTR—TT
  • SEQ ID NO: 115 (tr_E2JAJ2_Basidiomycota_Neolentinus_lepideus) SPIYGQC GGTGWTGATT CASGSTCVFS NPYYSQCLPG A-TTTTTSPQ PTTTTT TTTTN SGGGNPTTTT SAPGSTSTPD -AGPFVGYTL YLSPYYAAEV QA-AAGNITD
  • CAAAASNGEF SIANNGLANY ETYIDQLAAQ IQQYPDVRVV AVIEPDSLAN LVTNLNVAKC
  • IVVYDLPDRD CAALASNGEF SIANNGLANY KNYIDQLVAQ IKKYPDVRVV AVIEPDSLAN
  • VYKNAGSPAA LRGLATNVAN YNAWSISTCP
  • SYTQGDSNCD EKRYINALAP LLKAQGFPDA
  • VYKNAGSPAA LRGLATNVAN YNAWSISTCP
  • SYTQGDSVCD EKRYINALAP LLKAQGFPDA
  • VYKDAGSPAA LRGLATNVAN YNAWSISTCP
  • SYTQGDQNCD EKRYINALAP LLKAQGFPDA
  • VKTTSSTSVG TSSATTSTTT TPTTTTTTTTTTTT ASTTATTTAA ASGPFSGYQL

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Abstract

L'invention concerne des enzymes destinées à être utilisées dans la production de biocarburants. Certains aspects de la présente invention concernent des cellulases fongiques ancestrales. Les enzymes cellulases sont utiles pour la production d'éthanol cellulosique pour biocarburants. Dans certains modes de réalisation, les cellulases ancestrales peuvent être utilisées pour l'hydrolyse de polymères glucidiques qui comprennent de la cellulose. Certains aspects de la présente invention concernent des micro-organismes qui expriment une cellulase ancestrale. Les micro-organismes sont utiles pour la production d'éthanol cellulosique pour biocarburants. Dans certains modes de réalisation, les micro-organismes peuvent être utilisées pour l'hydrolyse et/ou la fermentation de la cellulose.
PCT/US2013/039306 2012-05-02 2013-05-02 Enzymes de production de biocarburants et leurs utilisations WO2013166312A1 (fr)

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US61/641,786 2012-05-02
US201261651199P 2012-05-24 2012-05-24
US61/651,199 2012-05-24

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015042543A3 (fr) * 2013-09-20 2015-05-14 The Trustees Of Columbia University In The City Of New York Enzymes de production de biocarburant et leurs utilisations
WO2017151957A1 (fr) * 2016-03-02 2017-09-08 Novozymes A/S Variants de cellobiohydrolase et polynucléotides codant pour ces derniers
US11286508B2 (en) * 2017-12-19 2022-03-29 Universidad Del País Vasco Ancestral cellulases and uses thereof

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US6127160A (en) * 1996-03-14 2000-10-03 Japan As Represented By Director General Of Agency Of Industrial Science And Technology Protein having cellulase activities and process for producing the same
US20030082595A1 (en) * 2001-08-03 2003-05-01 Bo Jiang Nucleic acids of aspergillus fumigatus encoding industrial enzymes and methods of use
WO2006074005A2 (fr) * 2004-12-30 2006-07-13 Genencor International, Inc. Nouveaux variants de cellulases d'hypocrea jecorina cbh2
US20060218671A1 (en) * 2005-01-06 2006-09-28 Novozymes, Inc. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
US20090325240A1 (en) * 2008-02-29 2009-12-31 Henry Daniell Production and use of plant degrading materials
US20110159544A1 (en) * 2009-12-30 2011-06-30 Roal Oy Method for treating cellulosic material and CBHII/CEL6A enzymes useful therein

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6127160A (en) * 1996-03-14 2000-10-03 Japan As Represented By Director General Of Agency Of Industrial Science And Technology Protein having cellulase activities and process for producing the same
US20030082595A1 (en) * 2001-08-03 2003-05-01 Bo Jiang Nucleic acids of aspergillus fumigatus encoding industrial enzymes and methods of use
WO2006074005A2 (fr) * 2004-12-30 2006-07-13 Genencor International, Inc. Nouveaux variants de cellulases d'hypocrea jecorina cbh2
US20060205042A1 (en) * 2004-12-30 2006-09-14 Wolfgang Aehle Novel variant hypocrea jecorina CBH2 cellulases
US20060218671A1 (en) * 2005-01-06 2006-09-28 Novozymes, Inc. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same
US20090325240A1 (en) * 2008-02-29 2009-12-31 Henry Daniell Production and use of plant degrading materials
US20110159544A1 (en) * 2009-12-30 2011-06-30 Roal Oy Method for treating cellulosic material and CBHII/CEL6A enzymes useful therein

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015042543A3 (fr) * 2013-09-20 2015-05-14 The Trustees Of Columbia University In The City Of New York Enzymes de production de biocarburant et leurs utilisations
WO2017151957A1 (fr) * 2016-03-02 2017-09-08 Novozymes A/S Variants de cellobiohydrolase et polynucléotides codant pour ces derniers
CN109415712A (zh) * 2016-03-02 2019-03-01 诺维信公司 纤维二糖水解酶变体和编码它们的多核苷酸
US10738293B2 (en) 2016-03-02 2020-08-11 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
US11053489B2 (en) 2016-03-02 2021-07-06 Novozymes A/S Cellobiohydrolase variants and polynucleotides encoding same
US11286508B2 (en) * 2017-12-19 2022-03-29 Universidad Del País Vasco Ancestral cellulases and uses thereof

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