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WO2018106656A1 - Enzymes de lpmo tronqués et leur utilisation - Google Patents

Enzymes de lpmo tronqués et leur utilisation Download PDF

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Publication number
WO2018106656A1
WO2018106656A1 PCT/US2017/064651 US2017064651W WO2018106656A1 WO 2018106656 A1 WO2018106656 A1 WO 2018106656A1 US 2017064651 W US2017064651 W US 2017064651W WO 2018106656 A1 WO2018106656 A1 WO 2018106656A1
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Prior art keywords
lpmo
native
protein
linker
amino acid
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PCT/US2017/064651
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English (en)
Inventor
Anna May LAM
Chuanbin Liu
Daniel Esteban TORRES PAZMINO
Nicholai Rainsong DOUGLAS
Steven Sungjin KIM
Thijs Kaper
Saeid KARKEHABADI
Henrik Hansson
Mats Sandgren
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Danisco Us Inc
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Publication of WO2018106656A1 publication Critical patent/WO2018106656A1/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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/99—Miscellaneous (1.14.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source

Definitions

  • the present disclosure generally pertains to truncated lytic polysaccharide mono- oxygenase (LPMO) enzymes lacking a carbohydrate-binding module (CBM). Nucleic acids encoding these enzymes, compositions comprising these enzymes, and methods of producing and using these enzymes, for example, also pertain to the present disclosure.
  • LPMO truncated lytic polysaccharide mono- oxygenase
  • CBM carbohydrate-binding module
  • Cellulose and hemicellulose are the most abundant plant materials produced by photosynthesis. They can be degraded and used as an energy source by numerous microorganisms, including bacteria, yeast and fungi, that produce extracellular enzymes capable of hydrolysis of the polymeric substrates to monomeric sugars (Aro et al., 2001 , J. Biol. Chem. 276:24309-24314). As the limits of non-renewable resources approach, the potential of cellulose to become a major renewable energy resource is enormous (Krishna et al., 2001 , Bioresource Tech. 77: 193-196).
  • cellulosic material for biofuel production include the ready availability of large amounts of feedstock (e.g., lignocellulosic feedstock), avoiding burning or land filling otherwise suitable feedstock materials, and that the end biofuel product itself (e.g., ethanol) is cleaner to use.
  • feedstock e.g., lignocellulosic feedstock
  • end biofuel product e.g., ethanol
  • Wood, agricultural residues, herbaceous crops, and municipal solid wastes, for example, can serve as cellulosic feedstocks for biofuel production.
  • Processes of converting cellulosic feedstock to biofuel can entail enzymatic saccharification of the feedstock to sugars, which may then be fermented into biofuel with a suitable microorganism.
  • New and improved enzymes and related compositions have recently been developed, making saccharification of cellulosic material more efficient. Nevertheless, further improvements in the performance of saccharification enzymes are desired for producing sugars for use in fermentation and other purposes.
  • the present disclosure concerns a non-native lytic
  • polysaccharide monooxygenase (LPMO) protein comprising: an LPMO catalytic domain, and a linker or linker portion located C-terminal to the catalytic domain, wherein the linker or linker portion comprises an amino acid sequence that is (i) at least 12 residues in length and (ii) at least about 80% identical to an amino acid sequence located within the first 40 amino acid residues of a native LPMO linker or cellulase linker, wherein the non-native LPMO protein does not comprise a carbohydrate-binding module (CBM).
  • CBM carbohydrate-binding module
  • the present disclosure concerns a polynucleotide comprising a nucleotide sequence encoding a non-native LPMO protein as disclosed herein.
  • Another embodiment concerns a cell comprising such a polynucleotide.
  • the present disclosure concerns a method of producing a non-native lytic polysaccharide monooxygenase (LPMO) protein, the method
  • the present disclosure concerns a composition
  • the present disclosure concerns a method of hydrolyzing a cellulosic substrate, the method comprising contacting the substrate with a
  • composition comprising a non-native LPMO protein as disclosed herein, and at least one cellulase, under aqueous conditions.
  • the present disclosure concerns a composition
  • a composition comprising: (i) a first lytic polysaccharide monooxygenase (LPMO) protein comprising an LPMO catalytic domain, wherein the first LPMO protein does not comprise a carbohydrate-binding module (CBM), and (ii) a second LPMO protein comprising an LPMO catalytic domain, a linker, and a CBM.
  • LPMO lytic polysaccharide monooxygenase
  • CBM carbohydrate-binding module
  • Another embodiment disclosed herein concerns a method of hydrolyzing a cellulosic substrate, the method comprising contacting the substrate with the foregoing composition under aqueous conditions.
  • FIGs. 1 A and 1 B depict the genomic DNA encoding full-length wild type HjGH61A (FIG. 1 A), and the protein itself (FIG. 1 B).
  • FIGs. 1 A-B also show the positions of the primer pairs for preparing sequences encoding truncated HjGH61A proteins
  • HjGH61AwoCBM_1 SEQ ID NO:7) and HjGH61AwoCBM_2 (SEQ ID NO:8).
  • CBM carbohydrate-binding module; gDNA, genomic DNA.
  • FIGs. 2A-C each provide 4-12% Bis-Tris SDS-PAGE gels run in MOPS buffer.
  • FIG. 2A shows heterologous expression levels of mature HjGH61 AwoCBM_1 ,
  • HjGH61AwoCBM_2 HjGH61AwoCBM_2, and wild type HjGH61A.
  • HjGH61AwoCBM_2 As two bands, it was initially believed that these bands represent two different N-linked glycoforms. However, when HjGH61AwoCBM_2 expression samples were treated with endoglycosidase H, which cleaves saccharides from /V-linked glycoproteins, both bands shifted down instead of consolidating into one band. It is possible that the two bands represent differential O-linked glycosylation on the 25-mer linker of mature HjGH61 AwoCBM_2.
  • FIG. 2B shows heterologous expression levels of mature StGH61woCBM_1 , StGH61woCBM_2, and wild type StGH61 .
  • FIG. 2C shows heterologous expression levels of mature TsGH61 woCBM_1 .
  • FIG. 3 shows an alignment of the linkers (approx.) of HjGH61A, CtGH61 , StGH61 and TsGH61 (listed, respectively, as “Hj", “Ct", “St” and “Ts”).
  • the bold-underlined subsequences indicate a conserved region within these linkers, which is represented herein by SEQ ID NO:44.
  • the underlined subsequences indicate a longer conserved region within these linkers, which is represented herein by SEQ ID NO:45.
  • An asterisk ( * ) indicates positions having a single, fully conserved residue.
  • a colon (:) indicates conservation between residues having strongly similar properties.
  • a period (.) indicates conservation between residues having weakly similar properties.
  • FIGs. 4A-B show an alignment of full-length wild type LPMOs HjGH61A (SEQ ID NO:2), CtGH61 (SEQ ID NO:24), StGH61 (SEQ ID NO:30) and TsGH61 (SEQ ID NO:36) (listed, respectively, as “Hj", “Ct”, “St” and “Ts") along with mature wild type LPMOs TtGH61 E (SEQ ID NO:41 ), HjGH61 B (SEQ ID NO:42) and TaGH61 A (SEQ ID NO:43) (listed, respectively, as “Tt", "HjB” and “Ta”). While HjGH61A, CtGH61 , StGH61 and TsGH61 each have a linker followed by a CBM, TtGH61 E, HjGH61 B and TaGH61 A do not have a linker or CBM.
  • FIGs. 5 and 6 show certain alignments.
  • FIG. 5 shows an alignment of the catalytic domains (approx.) of HjGH61A, CtGH61 , StGH61 and TsGH61 (listed, respectively, as “Hj”, “Ct”, “St” and “Ts”).
  • FIG. 6 shows an alignment of the catalytic domains (approx.) of HjGH61A, CtGH61 , StGH61 , TsGH61 and MtGH61 (listed, respectively, as “Hj", “Ct”, “St”, “Ts” and “Mt”).
  • an asterisk ( * ) indicates positions having a single, fully conserved residue
  • a colon (:) indicates conservation between residues having strongly similar properties
  • a period (.) indicates conservation between residues having weakly similar properties.
  • FIGs. 7A-B show heterologous expression levels of mature MtGH61woCBM_1 , MtGH61 woCBM_2, and wild type MtGH61 .
  • FIG. 8 shows a 24- and 48-hour dose response for wild type HjGH61 A and HjGH61AwoCBM_2 in saccharification reactions comprising DUET enzyme cocktail and 15% dry solids substrate (DACS, dilute ammonia-pretreated corn stover).
  • DAS dry solids substrate
  • FIG. 9 shows 48- and 72-hour saccharification reactions comprising DUET enzyme cocktail and 15% dry solids substrate (DACS), with the addition of either or both of wild type HjGH61A and HjGH61AwoCBM_2.
  • DAS dry solids substrate
  • FIGs. 10A-B show Avicel® binding assays using HjGH61AwoCBM_2 and wild type HjGH61A (FIG. 10A), and StGH61woCBM_2 and wild type StGH61 (FIG. 10B).
  • FIG. 10A-B show Avicel® binding assays using HjGH61AwoCBM_2 and wild type HjGH61A (FIG. 10A), and StGH61woCBM_2 and wild type StGH61 (FIG. 10B).
  • FIG. 1 1 shows glucose yields of saccharification reactions over time, as disclosed in Example 5.2 below.
  • circles solid line (100% full-length enzymes, 18% solids); triangles, solid line (100% truncated [ACBM] enzymes, 18% solids); X's, solid line (50:50 mixture of full-length and truncated [ACBM] enzymes, 18% solids); circles, dashed line (100% full-length enzymes, 10% solids); triangles, dashed line (100% truncated [ACBM] enzymes, 10% solids); X's, dashed line (50:50 mixture of full-length and truncated [ACBM] enzymes, 18% solids).
  • FIG. 12 shows glucose, xylose and arabinose yields (g/L) of 120-hour
  • FIGs. 13A-B show glucose, xylose and arabinose yields (g/L) of 120-hour (FIG.
  • the ratios indicated on the x-axis refer to the ratio of full-length HjGH61 A to truncated (ACBM) HjGH61 A in the HjGH61 A component of the enzyme mixture used.
  • FIG. 14 shows glucose, xylose and arabinose yields (g/L) of 48-hour
  • the x- axis refers to the GH61 component(s) (full-length and/or truncated [ACBM]) of the enzyme mixture used.
  • StGH61 full-length wild type, Sporotrichum thermophile.
  • Catalytic domain (approx.): residues 20-245 of SEQ ID NO:30.
  • TsGH61 full-length wild type, Trichoderma saturni. Mature
  • Catalytic domain (approx.): residues 22-248 of SEQ ID NO:36.
  • Mature form 38 predicted to be amino acid residues 22-313 of SEQ ID NO:38. 37 a (313 aa)
  • Mature form 40 predicted to be amino acid residues 22-252 of SEQ ID NO:40. 39 a (252 aa)
  • TtGH61 E mature wild type, Thielavia terrestris NRRL 8126.
  • HjGH61 B mature wild type, H. jecorina. (230 aa)
  • TaGH61A mature wild type, Thermoascus aurantiacus. (229 aa)
  • MtGH61 full-length wild type, Myceliophthora thermophila.
  • Catalytic domain (approx.): residues 19-241 of SEQ ID NO:46.
  • LPMO lytic polysaccharide mono-oxygenase
  • An LPMO enzyme can refer to (i) an auxiliary activity family 9 (AA9) protein, AA9 (formerly GH61 ) protein, EG4 protein, CEL61A protein, and/or glycoside hydrolase family 61 (GH61 ) protein, (ii) an auxiliary activity family 10 (AA10) protein and/or CBM33 protein, (iii) an auxiliary activity family 1 1 (AA1 1 ) protein, and/or an auxiliary activity family 13 (AA13) protein.
  • Current LPMO functional and classification details are as disclosed in Quinlan et al. (201 1 , Proc. Natl. Acad. Sci.
  • An LPMO herein is a copper-dependent enzyme that can enhance enzymatic degradation of cellulose by cellulase enzymes, and is believed to do so, at least in part, by oxidatively cleaving glycosidic bonds (using molecular oxygen) on the surface of cellulose without requiring separation of a glucan chain.
  • LPMO enzymes of the AA9 variety were originally classified as glycoside hydrolase family members based on measurement of very weak endo-1 ,4-beta-D-glucanase activity in one family member.
  • GH61 is used in name only and preferably not in particular reference to glycoside hydrolase function.
  • LPMO enzymes in full-length form typically contain (in the N-terminal to C-terminal direction) a signal peptide (cleaved away from mature form of an LPMO), a catalytic domain, linker, and carbohydrate-binding module (CBM); some native LPMO enzymes lack linker and CBM regions.
  • LPMO catalytic domain refers to the domain of an LPMO enzyme that provides its activity of enhancing the enzymatic degradation of cellulose by cellulase enzymes.
  • Modified LPMO proteins of interest herein comprise an LPMO catalytic domain in the N-terminal portion thereof.
  • suitable LPMO catalytic domains are within, approximately, amino acid residues 22-248 of SEQ ID NO:2 (HjGH61A), 20-245 of SEQ ID NO:24 (CtGH61 ), 20-245 of SEQ ID NO:30 (StGH61 ), 22- 248 of SEQ ID NO:36 (TsGH61 ), and 19-241 of SEQ ID NO:46 (MtGH61 ).
  • an LPMO catalytic domain comprises an amino acid sequence that is at least 30% identical to any of these particular catalytic domains.
  • linker refers to the amino acid sequence, typically about 40-60 residues in length, that joins the catalytic domain and CBM of a full-length LPMO, and is typically glycosylated.
  • a linker herein can be from a cellulase, in which case it joins the cellulase catalytic domain with the CBM of the cellulase in its full-length form.
  • a linker as referenced herein, or portion thereof, can be from an LPMO or cellulase enzyme, for example.
  • linkers herein include amino acid residues (approx.) 249-306 of SEQ ID NO:2 (HjGH61A), 246-303 of SEQ ID NO:24 (CtGH61 ), 246-309 of SEQ ID NO:30 (StGH61 ), and 249-313 of SEQ ID NO:36 (TsGH61 ).
  • a linker or portion thereof herein can, for example, comprise an amino acid sequence that is at least about 80% identical to SEQ ID NO:44 or 45.
  • Modified LPMO proteins of interest herein can comprise an entire linker or a portion thereof (detailed further below), where such linker sequence is located C-terminal to the LPMO catalytic domain.
  • CBM carbohydrate-binding module
  • carbohydrate-binding domain refers to a motif that can bind to a carbohydrate such as cellulose.
  • Modified LPMO proteins of interest herein do not comprise a CBM by virtue of a deletion/C-terminal truncation (e.g., CBM completely deleted); thus, modified LPMO proteins herein can optionally be referred to as C-terminally truncated LPMO proteins.
  • CBM's were previously referred to in the art as cellulose-binding modules.
  • CBM's herein for reference purposes include amino acid residues (approx.) 307-344 of SEQ ID NO:2 (HjGH61A), 304-336 of SEQ ID NO:24 (CtGH61 ), 310-342 of SEQ ID NO:30 (StGH61 ), and 314-346 of SEQ ID NO:36 (TsGH61 ).
  • a modified LPMO either completely lacks any amino acid sequence of a CBM, or has only a portion of a CBM (typically N-terminal portion thereof), which portion does not bind to carbohydrate.
  • transfermentable sugar(s) and like terms herein refer to oligosaccharides and monosaccharides that can be used as a carbon source by a microorganism in a fermentation process.
  • cellulosic and like terms herein refer to a composition comprising cellulose and typically additional components that may include hemicellulose and lignin.
  • lignocellulosic and like terms refer to a composition comprising both lignin and cellulose. Lignocellulosic material may also comprise hemicellulose.
  • biomass refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides, and/or monosaccharides.
  • Cellulosic biomass may also comprise additional components, such as protein and/or lipid.
  • Cellulosic biomass may be derived from a single source, or can comprise a mixture derived from more than one source; for example, cellulosic biomass could comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves.
  • Cellulosic biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste.
  • Further examples of biomass include corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum or soy cellulosic plant material, cellulosic components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes (woody plant cellulosic components), vegetables, fruits, flowers, and animal manure.
  • pretreated biomass and like terms herein refer to biomass that has been subjected to thermal, physical and/or chemical pretreatment to increase the availability of polysaccharides in the biomass to saccharification enzymes.
  • saccharification and like terms herein refer to the production of sugars (e.g., glucose, and optionally also xylose and/or arabinose) from polysaccharides. Such sugars typically are fermentable. Saccharification can be conducted, for example, by contacting a saccharification enzyme(s) with cellulosic or lignocellulosic material (typically that has been pretreated prior to enzymatic treatment). Such enzymatic saccharification typically is conducted in a slurry comprising at least water and insoluble cellulosic biomass. Saccharification can be monitored, for example, by measuring production of glucose, and optionally also xylose and/or arabinose.
  • biomass hydrolysate and like terms herein refer to the product resulting from saccharification of biomass.
  • the biomass used to prepare hydrolysate can be pretreated prior to saccharification.
  • the terms "clarified hydrolysate”, “clarified cellulosic hydrolysate” and the like herein refer to a biomass hydrolysate that has been processed to remove solids. Hydrolysates comprise dissolved sugars resulting from saccharification of biomass.
  • sacharification enzyme and like terms herein refer to an enzyme that can catalyze conversion of a polysaccharide component of biomass to fermentable sugars. Typically, the enzyme is more effective when the biomass is pretreated.
  • saccharification enzymes herein include cellulases.
  • cellulase refers to enzymes having endocellulase activity (EC 3.2.1 .4), exocellulase activity (EC 3.2.1 .91 ), or cellobiase activity (EC 3.2.1.21 ), and include exoglucanases, exocellobiohydrolases (cellobiohydrolases; e.g., CBH1 , CBH2), endoglucanases (e.g., EG1 , EG2), and/or beta- glucosidases, for example. These types of cellulase enzymes are capable of
  • a cellulase herein is typically of microbial origin (e.g., bacterial or fungal).
  • enzyme cocktail refers to a composition (typically aqueous, but can also be dry) comprising a heterogenous combination of two or more saccharification enzymes.
  • An enzyme cocktail typically can comprise one or more cellulases, and can in some aspects include one or more of a debranching enzyme, hemicellulase,
  • pentosanase xylanolytic enzyme, exoxylanase, endoxylanase (e.g., XynD), glucanase, exoglucanase, endo-beta-1 ,4-xylanase, exo-beta-1 ,4-xylosidase, L-alpha- arabinofuranosidase, endo-alpha-1 ,5-arabinanase, glucuronidase, alpha-glucuronidase, mannanase, endo-beta-1 ,4-mannanase, exo-beta-1 ,4-mannosidase, alpha- galactosidase, endo-galactanase, xylosidase, acetyl xylan esterase, glycosidase, beta- 1 ,4-glycanase, pectinase, polygalacturona
  • An enzyme cocktail in some aspects can be produced, at least in part, using a fungus (e.g., Trichoderma reesei) that expresses one or more of enzyme components of the cocktail.
  • a fungus e.g., Trichoderma reesei
  • enzyme cocktails that are, or have been, commercially available include SPEZYME CP, Accellerase®-1000, -1500, -DUET and -TRIO (DuPont), and Celluclast ® and Cellic CTec2® (Novozymes).
  • Examples of cocktails are disclosed in U.S. Patent Appl. Publ. Nos. 2014/0051 129, 2015/0176034 and 2010/0086981 , which are all incorporated herein by reference.
  • a mature protein is one that can pass through the cellular membrane of a cell such as a fungal cell.
  • a mature protein in some aspects results from post-translational removal (cleavage away) of a "signal sequence" (or “signal peptide") from the N-terminus of the protein's immature (preprocessed) form.
  • a signal sequence typically directs an immature protein to the cell membrane, and is removed from the protein during transit thereof through the membrane (i.e., during the protein secretion process).
  • Heterologous expression herein of a mature protein can employ a signal sequence, in which case the likely goal is secretion of the protein to the surrounding media.
  • heterologous expression can employ expressing a protein designed to already lack its signal sequence (a start methionine is typically added to the N-terminus in such embodiments); such mature protein expression typically entails lysing cells to release the protein, since it is not secreted.
  • a signal sequence herein can either be native or heterologous with respect to the protein with which it is optionally employed.
  • An LPMO protein e.g., C-terminally truncated form
  • the terms "polynucleotide”, “polynucleotide sequence”, “nucleic acid molecule” and the like are used interchangeably herein. These terms encompass nucleotide sequences and the like.
  • a polynucleotide may be a polymer of DNA or RNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases.
  • a polynucleotide may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.
  • RNA refers to a DNA polynucleotide sequence that expresses an RNA (RNA is transcribed from the DNA polynucleotide sequence) from a coding region, which RNA can be a messenger RNA (encoding a protein) or a non- protein-coding RNA.
  • a gene may refer to the coding region alone, or may include regulatory sequences upstream and/or downstream to the coding region (e.g., promoters, 5'-untranslated regions, 3'-transcription terminator regions).
  • a coding region encoding a protein can alternatively be referred to herein as an "open reading frame" (ORF).
  • a gene that is "native” or “endogenous” refers to a gene as found in nature with its own regulatory sequences; such a gene is located in its natural location in the genome of a host cell.
  • a “chimeric” gene refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature (i.e., the regulatory and coding regions are heterologous with each other). Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.
  • a “foreign” or “heterologous” gene can refer to a gene that is introduced into the host organism by gene transfer.
  • Foreign/heterologous genes can comprise native genes inserted into a non-native organism, native genes introduced into a new location within the native host, or chimeric genes.
  • Polynucleotide sequences in certain embodiments disclosed herein are heterologous.
  • a “transgene” is a gene that has been introduced into the genome by a gene delivery procedure (e.g., transformation).
  • a "codon- optimized" open reading frame has its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.
  • polypeptide is defined as a chain of amino acid residues, usually having a defined sequence.
  • polypeptide is interchangeable with the terms "peptides" and "proteins".
  • Typical amino acids contained in polypeptides herein include (respective three- and one-letter codes shown parenthetically): alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid (Glu, E), glutamine (Gin, Q), glycine (Gly, G), histidine (His, H), isoleucine (lie, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan
  • codon-optimized refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, herein refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to optimize the production of the polypeptide encoded by the DNA without altering the sequence of the polypeptide.
  • heterologous means not naturally found in the location of interest.
  • a heterologous gene can be one that is not naturally found in a host organism, but that is introduced into the host organism by gene transfer.
  • a nucleic acid molecule that is present in a chimeric gene can be characterized as being heterologous, as such a nucleic acid molecule is not naturally associated with the other segments of the chimeric gene (e.g., a promoter can be heterologous to a coding sequence).
  • a "non-native" amino acid sequence or polynucleotide sequence comprised in a cell or organism herein does not occur in a native (natural) counterpart of such cell or organism. Such an amino acid sequence or polynucleotide sequence can also be referred to as being heterologous to the cell or organism.
  • a non-native LPMO protein herein does not occur in nature, typically since it is a C-terminally truncated LPMO.
  • regulatory sequences refer to nucleotide sequences located upstream of a gene's transcription start site (e.g., promoter), 5' untranslated regions, introns, and 3' non-coding regions, and which may influence the transcription,
  • Regulatory sequences herein may include promoters, enhancers, silencers, 5' untranslated leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, stem-loop structures, and other elements involved in regulation of gene expression.
  • a "promoter” as used herein refers to a DNA sequence capable of controlling the transcription of RNA from a gene. In general, a promoter sequence is upstream of the transcription start site of a gene. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments.
  • Promoters that cause a gene to be expressed in a cell at most times under all circumstances are commonly referred to as "constitutive promoters".
  • a promoter may alternatively be inducible.
  • One or more promoters herein may be heterologous to a coding region herein.
  • a "strong promoter” as used herein refers to a promoter that can direct a relatively large number of productive initiations per unit time, and/or is a promoter driving a higher level of gene transcription than the average transcription level of the genes in a cell.
  • 3' non-coding sequence refers to DNA sequences located downstream of a coding sequence. This includes polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • upstream and downstream as used herein with respect to polynucleotides refer to "5' of” and “3' of”, respectively.
  • RNA e.g., mRNA or a non-protein-coding RNA
  • expression of a coding region of a polynucleotide sequence can be up-regulated or down-regulated in certain embodiments.
  • operably linked refers to the association of two or more nucleic acid sequences such that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence. That is, the coding sequence is under the transcriptional control of the promoter.
  • a coding sequence can be operably linked to one (e.g., promoter) or more (e.g., promoter and terminator) regulatory sequences, for example.
  • nucleic acid molecule when used herein to characterize a DNA sequence such as a plasmid, vector, or construct refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis and/or by manipulation of isolated segments of nucleic acids by genetic engineering techniques.
  • transformation refers to the transfer of a nucleic acid molecule into a host organism or host cell by any method.
  • a nucleic acid molecule that has been transformed into an organism/cell may be one that replicates autonomously in the organism/cell, or that integrates into the genome of the organism/cell, or that exists transiently in the cell without replicating or integrating.
  • Non-limiting examples of nucleic acid molecules suitable for transformation are disclosed herein, such as plasmids and linear DNA molecules.
  • Host organisms/cells herein containing a transforming nucleic acid sequence can be referred to as "transgenic”, “recombinant”, “transformed”,
  • sequence identity refers to the nucleic acid residues or amino acid residues in two sequences that are the same when aligned for maximum
  • percentage of sequence identity refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity.
  • T residues of the DNA sequence align with, and can be considered “identical” with, U residues of the RNA sequence.
  • percent complementarity For purposes of determining "percent complementarity" of first and second polynucleotides, one can obtain this by determining (i) the percent identity between the first polynucleotide and the complement sequence of the second polynucleotide (or vice versa), for example, and/or (ii) the percentage of bases between the first and second polynucleotides that would create canonical Watson and Crick base pairs.
  • Percent identity can be readily determined by any known method, including but not limited to those described in: 1 ) Computational Molecular Biology (Lesk, A.M., Ed.) Oxford University: NY (1988); 2) Biocomputing: Informatics and Genome Projects
  • Preferred methods for determining percent identity are designed to give the best match between the sequences tested. Methods of determining identity and similarity are codified in publicly available computer programs, for example. Sequence alignments and percent identity calculations can be performed using the MEGALIGN program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wl), for example. Multiple alignment of sequences can be performed, for example, using the Clustal method of alignment which encompasses several varieties of the algorithm including the Clustal V method of alignment (described by Higgins and Sharp, CABIOS. 5: 151 -153 (1989); Higgins, D.G. et al., Comput. Appl.
  • SAVED 5.
  • the Clustal W method of alignment can be used (described by Higgins and Sharp, CABIOS. 5:151 -153 (1989); Higgins, D.G. et al., Comput. Appl. Biosci. 8: 189-191 (1992); Thompson, J.D. et al, Nucleic Acids Research, 22 (22): 4673-4680, 1994) and found in the MEGALIGN v8.0 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.).
  • Various polypeptide amino acid sequences and polynucleotide sequences are disclosed herein as features of certain embodiments. Variants of these sequences that are at least about 70-85%, 85-90%, or 90%-95% identical to the sequences disclosed herein can be used or referenced.
  • a variant amino acid sequence or polynucleotide sequence can have at least 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% or 99% identity with a sequence disclosed herein.
  • the variant amino acid sequence or polynucleotide sequence has the same function/activity of the disclosed sequence, or at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the function/activity of the disclosed sequence.
  • Any polypeptide amino acid sequence disclosed herein not beginning with a methionine can typically further comprise at least a start-methionine at the N-terminus of the amino acid sequence.
  • any polypeptide amino acid sequence disclosed herein beginning with a methionine can optionally lack such a methionine residue.
  • isolated means a substance (or process) in a form or environment that does not occur in nature.
  • isolated substances include (1 ) any non-naturally occurring substance (e.g., a truncated LPMO enzyme herein), (2) any substance including, but not limited to, any host cell, enzyme, variant, nucleic acid, protein, peptide, cofactor, or carbohydrate/saccharide that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature (e.g., a truncated LPMO enzyme herein); or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated. It is believed that the embodiments (e.g., enzymes and reaction compositions) disclosed herein are only synthetic/man-made, and/or have properties that are not naturally occurring.
  • %(w/v) values of cellulosic substrates herein, when provided in an aqueous preparation such as a saccharification reaction are given as %(w/v).
  • percent dry solids in a reaction is based on the dry weight of the solids provided in a total volume of reaction.
  • aqueous conditions refer to a solution or mixture in which the solvent is at least about 60 wt% water, for example.
  • a saccharification reaction herein is performed under aqueous conditions.
  • the term "increased” as used herein can refer to a quantity or activity that is at least about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 50%, 100%, or 200% more than the quantity or activity for which the increased quantity or activity is being compared.
  • the terms “increased”, “elevated”, “enhanced”, “greater than”, “improved” and the like are used interchangeably herein. These terms can be used to characterize the "over-expression” or "up- regulation" of a polynucleotide encoding a protein, for example.
  • Non-native LPMO protein comprising: a lytic polysaccharide mono-oxygenase (LPMO) catalytic domain, and a linker located C-terminal to the catalytic domain, wherein the linker comprises an amino acid sequence that is (i) at least 12 residues in length and (ii) at least about 80% identical to an amino acid sequence located within the first 40 amino acid residues of a native LPMO linker or native cellulase linker, wherein the non-native LPMO protein does not comprise a carbohydrate-binding module (CBM).
  • CBM carbohydrate-binding module
  • non- native, C-terminally truncated LPMO protein are contemplated to include being able to recombinantly express it in a microorganism at suitably high levels, as compared to expressing an otherwise identical protein that only differs by not having such a linker.
  • the linker of a non-native LPMO herein comprises or consists of an amino acid sequence that can be derived from within the first 40 amino acid residues of a native LPMO linker or cellulase linker.
  • Such an amino acid sequence can be derived from within the first 40, 39, 38, 37, 36, 35, 34, 33, 32, 31 , 30, 29, 28, 27, 25, 24, 23, or 22 (or a range between any two of these values) amino acid residues of a native LPMO linker or cellulase linker, for example.
  • the "first" amino acid residues of a linker typically are those that are immediately C-terminally adjacent to the catalytic domain of an LPMO.
  • a linker of a native LPMO can be identified, for example, by aligning the amino acid sequence of the LPMO with the amino acid sequence(s) of an LPMO(s) in which catalytic domain and linker regions have previously been characterized, and/or with an LPMO(s) previously characterized to lack a linker region.
  • FIGs. 4A-B show an example of an alignment of native LPMO proteins, some having a linker region (followed by CBM) and some not having a linker and CBM. The portion of the alignment in FIGs. 4A-B in which all seven sequences align encompasses the approximate catalytic domains of the sequences. Similar alignments can be conducted accordingly to identify the
  • the linker of a non-native LPMO as presently disclosed can comprise or consist of an amino acid sequence that is (i) at least 12 residues in length and (ii) at least about 80% identical to an amino acid sequence located within the first 40 amino acid residues of a native LPMO linker or cellulase linker.
  • such an amino acid sequence is at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or a range between any two of these values) residues in length and/or at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, an amino acid sequence located within the first 40 amino acid residues of a native LPMO linker or cellulase linker.
  • the linker of a non-native LPMO as presently disclosed can comprise or consist of, for example, an amino acid sequence that is at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, SEQ ID NO:44.
  • Examples of such an amino acid sequence include residues 259-272 of SEQ ID NO:2, residues 256-269 of SEQ ID NO:24, residues 256-269 of SEQ ID NO:30, and residues 259-272 of SEQ ID NO:36.
  • the linker of a non-native LPMO can comprise or consist of an amino acid sequence that is at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, SEQ ID NO:45.
  • amino acid sequence examples include residues 253-272 of SEQ ID NO:2, residues 250-269 of SEQ ID NO:24, residues 250-
  • the linker of a non-native LPMO can comprise or consist of an amino acid sequence that is at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, residues 249-273 of SEQ ID NO:2, residues 246-270 of SEQ ID NO:24, residues 246-
  • a linker in some aspects can be a full-length linker of a native LPMO or cellulase (e.g., any of the linker regions specified in Table 1 ), while a linker in other aspects is not a full-length native LPMO linker or cellulase linker (and can optionally be characterized as a "linker portion" or "partial linker”).
  • the length of a linker herein can be 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35 residues or less, for instance.
  • the linker of a non-native LPMO comprises an amino acid sequence that (i) is located immediately C-terminal to the catalytic domain of a native LPMO or cellulase, or (ii) begins at the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, or fifteenth residue of a native LPMO or cellulase linker.
  • linker of a non-native LPMO herein is typically autologous to (native to) the catalytic domain of the non-native LPMO (i.e., catalytic domain and linker sequences are derived from the same native LPMO), it can be heterologous to the catalytic domain in some embodiments.
  • a non-native LPMO herein typically is not associated with a ribosome or component (protein and/or RNA) thereof.
  • the linker of a non-native LPMO herein can alternatively be characterized simply as an "amino acid sequence C-terminal to the catalytic domain" of the non-native LPMO (and like terminology), since a linker in typical embodiments herein is not actually linking a catalytic domain to a CBM (since the non-native LPMO does not have a CBM).
  • a non-native LPMO herein typically is derived from (corresponds to) a counterpart native LPMO that has a CBM.
  • a non-native LPMO protein herein comprises an LPMO catalytic domain.
  • an LPMO catalytic domain provides to an LPMO the ability to enhance enzymatic degradation of cellulose by cellulase enzymes. Methods of determining such activity herein can be as disclosed in U.S. Patent Appl. Publ. No. 2014/0127771 (e.g., para. 78 therein), for example, which is incorporated herein by reference.
  • LPMO enzymes are currently classified in three families of auxiliary enzymes in the Carbohydrate Active Enzymes (CAZy) database (www.cazy.org; Lombard et al., 2014, Nucleic Acids Res. 42:D490-D495; Levasseur et al., 2013, Biotechnology for Biofuels 6:41 ; each incorporated herein by reference).
  • CAZy Carbohydrate Active Enzymes
  • the AA9 and AA1 1 families of LPMO's are currently dominated by eukaryotic enzymes, with apparent substrate preferences for cellulose and chitin, respectively.
  • the AA10 family of LPMO's is currently dominated by bacterial enzymes, with some examples of cellulose and chitin specificity.
  • An LPMO catalytic domain herein can be a bacterial LPMO catalytic domain from an AA10 protein, for instance. Suitable examples thereof can be found, for example, in the CAZy database, including those with Gram-positive bacterial LPMO catalytic domains such as Bacillus, Streptomyces, Enterococcus, Lactobacillus, Lactococcus, Listeria, Cellulomonas, Oceanobacillus, Thermobifida, and Thermobispora LPMO catalytic domains, and Gram-negative bacterial LPMO catalytic domains such as
  • An LPMO catalytic domain herein can be a fungal (e.g., yeast or filamentous fungus) LPMO catalytic domain, for instance.
  • yeast LPMO catalytic domains include Candida, Kluyveromyces, Pichia, Saccharomyces,
  • filamentous fungal LPMO catalytic domains include Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Hypocrea, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,
  • Penicillium Phanerochaete, Piromyces, Podospora, Poitrasia, Pseudoplectania,
  • filamentous fungal LPMO catalytic domains include Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,
  • Chrysosporium lucknowense Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Cornyascus thermophilus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum,
  • Fusarium heterosporum Fusarium negundi, Fusarium oxysporum, Fusarium reticuiatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium
  • sporotrichioides Fusarium suiphureum, Fusarium toruiosum, Fusarium trichothecioides, Fusarium venenatum, Fusarium verticilliloides, Humicola grisea, Humicola insolens, Humicola lanuginosa, Hypocrea jecorina, Irpex lacteus, Mucor miehei, Myceliophthora fergusii, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Penicillium thomfi, Phanerochaete chrysosporium,
  • Thielavia achromatica Thielavia albomyces, Thielavia albopilosa
  • Thielavia australeinsis Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichophaea saccata, Trichoderma saturni and Trichoderma viride LPMO catalytic domains.
  • An LPMO catalytic domain is not a Neurospora (e.g., N. crassa), Podospora (e.g., P.
  • anserina or Lentinus (e.g., L. similis) LPMO catalytic domain in some aspects.
  • An LPMO catalytic domain can, in some aspects, be a catalytic domain of an AA9 enzyme, but not of an AA10, AA1 1 and/or AA13 enzyme.
  • an LPMO catalytic domain can be of an LPMO that, in native form, comprises (in the N-terminal to C-terminal direction) a catalytic domain, linker, and CBM; an LPMO catalytic domain in such aspects is not from an LPMO that, in native form, lacks linker and/or CBM regions.
  • an LPMO catalytic domain can (i) comprise or consist of an amino acid sequence that is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to, the catalytic domain of HjGH61A (residues 22-248 of SEQ ID NO:2), CtGH61 (residues 20-245 of SEQ ID NO:24), StGH61 (residues 20-245 of SEQ ID NO:30), TsGH61 (residues 22-248 of SEQ ID NO:36), or 19-241 of SEQ ID NO:46 (MtGH61 ), and (ii) have LPMO catalytic activity.
  • HjGH61A
  • An LPMO catalytic domain in some embodiments can comprise at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of, or all of, the fully conserved residues/positions (and optionally also the strongly conserved
  • residues/positions of some or all these particular catalytic domains (e.g., fully and/or strongly conserved residues/positions as indicated in the alignment of FIG. 5 or FIG. 6).
  • a non-native LPMO protein herein does not comprise a carbohydrate-binding module (CBM). While it is typical for a non-native LPMO protein as presently disclosed to completely lack any CBM amino acid sequence, a non-native LPMO protein can optionally comprise a portion (e.g., less than about 5-10 amino acid residues) of a CBM amino acid sequence that does not bind to carbohydrate (e.g., cellulose). Such a sequence portion does not function as a CBM, and thus is not considered to be a CBM, per se.
  • CBM carbohydrate-binding module
  • any amino acid sequence C-terminal to the linker or partial linker sequence of a non-native LPMO protein in certain embodiments has (i) less than about 35%, 30%, 25%, or 20% identity to an AA9 protein CBM, or to residues 307-344 of SEQ ID NO:2 (HjGH61A), 304-336 of SEQ ID NO:24 (CtGH61 ), 310-342 of SEQ ID NO:30 (StGH61 ), or 314-346 of SEQ ID NO:36 (TsGH61 ), and/or (ii) no carbohydrate-binding activity.
  • a non-native LPMO protein in certain embodiments can comprise or consist of an amino acid sequence that is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, or 100% identical to,
  • HjGH61AwoCBM_2 (SEQ ID NO: 10), CtGH61woCBM_2 (SEQ ID NO:26),
  • StGH61woCBM_2 (SEQ ID NO:32), TsGH61woCBM_2 (SEQ ID NO:38), or MtGH61woCBM_2 (SEQ ID NO:47), which are exemplified below as suitable non-native LPMO proteins herein (refer to Examples).
  • a non-native LPMO protein in certain embodiments has the same function/activity of its native counterpart LPMO (native counterpart has same catalytic domain), or at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 105%, 1 10%, or 1 15% of the
  • Such activity can be as measured under any saccharification reaction conditions disclosed herein (e.g., time, temperature, substrate and/or cellulase components/concentrations), for example.
  • non-native LPMO proteins herein can be comprised within (i.e., part of) a fusion protein.
  • a heterologous amino acid sequence can be linked at the N-terminus (i.e., next to the LPMO catalytic domain) and/or C-terminus (i.e., next to the linker or partial linker.
  • Such a heterologous amino acid sequence can be about, or at least about, 1 , 5, 10, 15, 20, 30, 40, or 50 residues long, for example.
  • a heterologous amino acid sequence can in some aspects comprise an epitope tag (N- or C-terminus) and/or a heterologous signal peptide (at N-terminus).
  • Some embodiments of the present disclosure concern a polynucleotide comprising a nucleotide sequence encoding a non-native LPMO protein as presently disclosed (e.g., a non-native C-terminally truncated LPMO protein comprising an LPMO catalytic domain and a linker located C-terminal to the catalytic domain, wherein the linker comprises an amino acid sequence that is (i) at least 12 residues in length and (ii) at least about 80% identical to an amino acid sequence located within the first 40 amino acid residues of a native LPMO linker or native cellulase linker, wherein the non-native LPMO protein does not comprise a CBM).
  • a non-native LPMO protein e.g., a non-native C-terminally truncated LPMO protein comprising an LPMO catalytic domain and a linker located C-terminal to the catalytic domain, wherein the linker comprises an amino acid
  • sequences are operably linked to the nucleotide sequence, and preferably a promoter sequence is included as a regulatory sequence.
  • a polynucleotide comprising a nucleotide sequence encoding a non-native LPMO protein herein can be a vector or construct useful for transferring a nucleotide sequence into a cell, for example.
  • a suitable vector/construct can be selected from a plasmid, yeast artificial chromosome (YAC), cosmid, phagemid, bacterial artificial chromosome (BAC), virus, or linear DNA (e.g., linear PCR product).
  • a polynucleotide sequence in some aspects can be capable of existing transiently (i.e., not integrated into the genome) or stably (i.e., integrated into the genome) in a cell.
  • a polynucleotide sequence in some aspects can comprise, or lack, one or more suitable marker sequences (e.g., selection or phenotype marker).
  • a polynucleotide sequence in certain embodiments can comprise one or more regulatory sequences operably linked to the nucleotide sequence encoding a non-native LPMO protein herein.
  • a nucleotide sequence encoding a non-native LPMO may be in operable linkage with a promoter sequence (e.g., a heterologous promoter).
  • a promoter sequence can be suitable for expression in a cell (e.g., bacterial cell such as E. coli or Bacillus; eukaryotic cell such as a fungus, yeast, insect, or mammalian cell) or in an in vitro protein expression system, for example. Examples of other suitable regulatory sequences are disclosed herein (e.g., transcription terminator sequences).
  • a cell comprising a polynucleotide sequence as presently disclosed.
  • a cell can be any type disclosed herein (e.g., bacterial cell such as E. coli or Bacillus [e.g., B. subtilis]; eukaryotic cell such as a fungus, yeast, insect, or mammalian cell).
  • a cell can express the non-native LPMO encoded by the polynucleotide sequence; in such embodiments, the nucleotide encoding the non-native LPMO is typically operably linked to a promoter that is functional in the cell.
  • the polynucleotide sequence exists transiently (i.e., not integrated into the genome) or stably (i.e., integrated into the genome) in the cell.
  • suitable cell types include any of those yeast and filamentous fungi disclosed elsewhere herein (e.g., above description or below Examples).
  • a suitable cell can be of a filamentous fungus such as Trichoderma (e.g., T. reesei, T. longibrachiatum, T. viride, T. koningii, T. harzianum), Penicillium, Humicola (e.g., H. insolens, H. grisea), Chrysosporium (e.g., C. lucknowense), Myceliophthora thermophila, Gliocladium, Fusarium, Neurospora, Hypocrea (e.g., H. jecorina),
  • Trichoderma e.g., T. reesei, T. longibrach
  • Emericella or Aspergillus (e.g., A. niger, A. awamori, A. aculeatus, A. nidulans).
  • Suitable filamentous fungal cells include T. reesei strains disclosed in U.S. Patent No. 5847276, U.S. Patent Appl. Publ. No. 2016/0122735, and Seiboth et al. (Trichoderma reesei: A Fungal Enzyme Producer for Cellulosic Biofuels, In Biofuel Production - Recent Developments and Prospects, ed. Bernardos, InTech, pp. 309-340, 201 1 ), which are each incorporated herein by reference.
  • a suitable cell can be of a yeast such as Saccharomyces (e.g., S. cerevisiae),
  • Debaryomyces hansenii or Debaryomyces polymorphus.
  • Some embodiments of the present disclosure concern a method of producing a non-native LPMO protein herein. Such a method can comprise the following steps:
  • an advantage of this method in typical embodiments is that the expression of the non- native LPMO protein in (b) is enhanced relative to the expression of a second protein that only differs from the non-native protein by comprising no more than 10 amino acid residues of a native LPMO linker or native cellulase linker.
  • a non-native C-terminally truncated LPMO as presently disclosed as compared to expressing a counterpart LPMO protein ("second protein”, "reference protein”, or the like) comprising little or no linker sequence.
  • the second protein only differs from the non-native LPMO by having no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues of a native LPMO linker or cellulase linker.
  • a limited amino acid sequence can be of any linker amino acid sequence as disclosed elsewhere herein, but just not meet the criteria of such a linker sequence.
  • the second protein can differ from the non- native LPMO by comprising, C-terminal to the LPMO catalytic domain, an amino acid sequence that is less than 80% identical to SEQ ID NO:44 or 45.
  • the second protein in some aspects has no amino acid residues of a linker (e.g., comprises only an LPMO catalytic domain, optionally with a heterologous sequence N-terminal to the catalytic domain).
  • a linker e.g., comprises only an LPMO catalytic domain, optionally with a heterologous sequence N-terminal to the catalytic domain.
  • Non-native LPMO protein in step (b) of an expression method herein is enhanced relative to the expression of a second protein.
  • Such enhanced expression of a non-native LPMO can be expression that is at least about 5%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 750%, or 1000% greater than the expression (if detectable and/or measurable) of the second protein.
  • a comparison in expression levels of a non-native LPMO and second protein can be made under the same or similar expression conditions. It should be understood that an expression comparison need not actually be performed to practice a method herein of expressing a non-native LPMO (i.e., the non-native LPMO has the capability of enhanced expression compared to a second protein herein).
  • Fungal cells for example, expressing a non-native LPMO protein herein can be cultured under conditions typically employed to culture fungal cells.
  • cells can be cultured in a standard medium containing physiological salts and nutrients, such as described in Pourquie et al. (Biochemistry and Genetics of Cellulose Degradation, eds. Aubert et al., Academic Press, pp. 71 -86, 1988) and llmen et al. (1997, Appl. Environ. Microbiol. 63: 1298-1306), which are each incorporated herein by reference.
  • fungal cells can be grown under the conditions disclosed in the below Examples, or under conditions in which one or more parameters (e.g., time,
  • a non-native LPMO product of an expression method herein can optionally be isolated.
  • a non-native LPMO produced in a cell culture herein is secreted into its surrounding medium (producing a culture supernatant containing the non-native LPMO) and may be enriched, purified, or isolated, for example, by removing one or more unwanted components from the medium.
  • a non- native LPMO may be produced in a cellular form necessitating recovery from a cell lysate.
  • Processes that may be used in an isolation step herein include, for example, filtration (e.g., ultra- or micro-filtration), centrifugation, density-gradient fractionation (e.g., density-gradient ultracentrifugation), chromatography (e.g., affinity, ion-exchange, hydrophobic interaction), evaporation, precipitation, and/or extraction.
  • a cell expressing a non-native LPMO is employed directly in a process that requires or benefits from LPMO activity.
  • a culture of, or a lysate of, a fungus that expresses a non-native LPMO herein and one or more cellulase enzymes can be used accordingly, such as in a saccharification process.
  • a supernatant of a culture of cells expressing a non-native LPMO can be collected and used as disclosed herein. Preparation of such a
  • supernatant can comprise one or more of the optional steps of killing the cells of a collected cell culture, filtering and/or centrifuging the collected cell culture to remove cells/cell debris, and/or subjecting the collected cell culture to ultrafiltration or other steps to enrich or concentrate the non-native LPMO.
  • compositions comprising a non-native LPMO protein as presently disclosed and at least one cellulase.
  • a composition is an example of a cellulolytic enzyme composition or cocktail.
  • a cellulolytic enzyme composition can further comprise one or more of the following proteins: a native LPMO, hemicellulase, esterase, expansin, laccase, ligninolytic enzyme, pectinase, peroxidase, protease, and swollenin.
  • the cellulase component(s) of a cellulolytic composition can include one or more of an endoglucanase, cellobiohydrolase, or beta-glucosidase.
  • a hemicellulase in some aspects can be one or more of an acetylmannan esterase, acetylxylan esterase, arabinanase, arabinofuranosidase, coumaric acid esterase, feruloyl esterase, galactosidase, glucuronidase, glucuronoyl esterase, mannanase, mannosidase, xylanase, and xylosidase.
  • Suitable examples of one or more of the foregoing enzymes are disclosed in U.S. Patent No. 9150842 and U.S. Patent Appl. Publ. Nos.
  • cellulytic enzyme compositions in which a non-native LPMO protein can be included are SPEZYME CP, Accellerase®-1000, -1500, -DUET and -TRIO, as well as those disclosed in U.S. Pat. Appl. Publ. No. 2014/0106408, which is incorporated herein by reference.
  • a cellulolytic enzyme composition can be in any form suitable for use in an application that requires or benefits from cellulolytic processing, for example.
  • a cellulolytic enzyme composition can be in the form of a fermentation broth (whole cell broth), cell culture supernatant, a cell lysate with or without cellular debris, a semi-purified or purified enzyme preparation, or a host cell that expresses some or all of the enzyme components.
  • a suitable cell e.g., any cell as disclosed herein, such as a filamentous fungal cell [e.g. T.
  • reesei or yeast cell
  • yeast cell that expresses a non-native LPMO herein and at least one cellulase, and optionally one or more other saccharification enzymes (such cellulase and other enzymes can be native and/or heterologous to the cell used for expression).
  • processes for producing these types of compositions are disclosed in U.S. Pat. Appl. Publ. No. 2014/0295475, which is incorporated herein by reference.
  • a cellulolytic enzyme composition can be in the form of a dry powder or granulate, or a liquid (e.g., stabilized liquid).
  • a stabilized liquid enzyme preparation can be prepared by adding stabilizers such as a sugar, sugar alcohol or another polyol, and/or lactic acid or another organic acid, for example.
  • a cellulolytic enzyme composition can be in the form of, and/or comprised within, a cellulolytic enzyme cocktail for saccharifying cellulosic biomass; a fabric care, detergent and/or textile treatment composition (e.g., comprising at least one surfactant); a human or animal food/feed composition (e.g., an additive) and/or processing composition; pulp/paper-processing composition; or grain processing (e.g., grain wet milling) composition, such as disclosed in U.S. Pat. No. 6017870 and U.S. Pat. Appl. Publ. Nos. 2015/0368594 and 2007/0028392, which are each incorporated herein by reference.
  • a fabric care, detergent and/or textile treatment composition e.g., comprising at least one surfactant
  • a human or animal food/feed composition e.g., an additive
  • processing composition e.g., grain wet milling
  • a cellulolytic enzyme composition in some aspects can comprise a
  • a saccharification reaction can characterize a saccharification reaction herein.
  • a saccharification reaction herein can comprise at least a cellulosic substrate, non-native LPMO protein herein, cellulase and water.
  • Some embodiments of the present disclosure concern a method for hydrolyzing a cellulosic substrate (saccharification). This method comprises contacting the cellulosic substrate with a composition comprising a non-native LPMO protein as disclosed herein and at least one cellulase, under aqueous conditions.
  • a hydrolysis method can comprise contacting a cellulose substrate with a cellulolytic enzyme composition as presently disclosed.
  • a cellulolytic enzyme composition can be provided as a cell-free composition (e.g., a cell culture supernatant or cell lysate), whereas in other embodiments, it can be provided as a cell that expresses the non-native LPMO and/or cellulase.
  • a hydrolysis method herein is typically practiced, at least in part, for converting cellulosic biomass to glucose and/or glucose-containing oligosaccharides (e.g., disaccharides), which optionally may in turn be used as a substrate in a suitable alcohol (e.g., ethanol) fermentation process.
  • a suitable alcohol e.g., ethanol
  • Another possible use of glucose and/or glucose-containing oligosaccharides herein include producing a syrup comprising the same.
  • suitable cellulosic substrates (feedstock) for a hydrolysis method herein include grass, switchgrass, cord grass, rye grass, reed canary grass, miscanthus, sugar-processing residues, sugarcane bagasse, sugarcane straw, sorghum, agricultural wastes, rice straw, rice hulls, barley straw, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn cobs, corn fiber, corn stover, soybean stover, forestry wastes, wood pulp, recycled wood pulp fiber, paper sludge, sawdust, hardwood, softwood, and combinations thereof.
  • a cellulose feedstock contains, on a dry weight percentage basis, (i) about 20-90%, 30-90%, 40-90%, 20-80%, 30-80%, or 40-80% cellulose, and/or (ii) at least about 10%, 1 1 %, or 12% lignin.
  • a cellulosic feedstock for hydrolysis can be pretreated.
  • Such pretreatment can comprise treating the substrate with elevated temperature and/or acid (e.g., a dilute acid such as dilute-sulfuric acid, a concentrated acid such as phosphoric acid or peracetic acid), alkali (e.g., ammonia, ammonium hydroxide, potassium
  • a pretreatment process in some aspects can be conducted using any pretreatment agent and/or procedure as disclosed in U.S. Pat. Nos.
  • Pretreated feedstock is typically processed after pretreatment by any of several steps, such as dilution with water, washing with water, buffering, filtration, centrifugation, or a combination of these processes, prior to enzymatic hydrolysis.
  • the pH of a pretreated feedstock preparation e.g., slurry
  • Pretreated feedstock can include dilute ammonia-pretreated corn stover (DACS, e.g., as disclosed in U.S. Pat. Appl. Publ. No.
  • dilute ammonia- pretreated switchgrass e.g., ibid.
  • dilute acid-pretreated corn stover PCS, e.g., as disclosed in Schell et al., 2003
  • PASC phosphoric-acid swollen cellulose
  • a hydrolysis method herein is typically performed at a pH and temperature that are at or near optimum for the saccharification enzymes used therein.
  • enzymatic hydrolysis may be carried out at about 30-60 °C, or any temperature therebetween (e.g., 30, 33, 35, 40, 45, 50, 55, 60 °C).
  • the pH of a hydrolysis reaction can be about 4.0-7.5, or any pH therebetween (e.g., 4.0, 5.0, 6.0, 6.5, 7.0, 7.5, 4.0-6.0).
  • a hydrolysis method can be carried out for a time period of about 1 to 120 hours, or any time therebetween.
  • a hydrolysis reaction can be held for about 4, 6, 8, 12, 24, 30, 36, 42, 48, 54, 60, 66, 72, 84, 96, 108, 120, 132, 144, 12-24, 12-48, 12-72, 24-48, 24-72, or 48-72 hours.
  • the initial percent dry solids (% w/v) of cellulose substrate (e.g., pretreated cellulose feedstock) in a hydrolysis reaction herein can be about, or at least about, 1 %, 5%, 10%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 10-20%, 10- 25%, 15-18%, 15-20%, or 15-25%, for example.
  • the combined dosage of all saccharification enzymes in a hydrolysis reaction herein can be, for example, about 0.001 to about 100 mg enzyme protein per gram cellulose (or per gram cellulose plus xylan) in the reaction, or any amount therebetween (e.g., about, or at least about, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, 25, 30, 5-15, 10- 15, or 10-20 mg enzyme protein per gram cellulose or cellulose+xylan).
  • the weight percentage of a non-native LPMO protein of the enzyme protein added to a hydrolysis reaction can be about, or at least about, 5%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 10-20%, 12-18%, 13-17%, or 14-16%, for example.
  • a hydrolysis reaction herein can be run by batch mode and/or continuous mode, for instance.
  • a hydrolysis reaction can be agitated, unmixed, or a combination thereof.
  • a hydrolysis reaction herein can comprise one or more of the conditions and/or saccharification schemes disclosed in Viikari et al. (2007, Adv.
  • Glucose and/or glucose-containing oligosaccharides (e.g., disaccharides) produced by a hydrolysis method herein can optionally be used as a substrate in a suitable alcohol (e.g., ethanol) fermentation process.
  • a suitable alcohol e.g., ethanol
  • a method of producing alcohol is further disclosed, which can comprise, for example, culturing a suitable microbial cell/biocatalyst (e.g., ethanologen) in a medium comprising the foregoing glucose and/or glucose-containing oligosaccharide substrate(s), whereby the
  • Suitable microbial cells for such an alcohol fermentation/production process include yeast (e.g., Saccharomyces such as S. cerevisiae), bacteria (e.g., Zymomonas such as Z. mobilis, Zymobacter), and recombinant forms thereof, for example.
  • yeast e.g., Saccharomyces such as S. cerevisiae
  • bacteria e.g., Zymomonas such as Z. mobilis, Zymobacter
  • recombinant forms thereof for example.
  • Some examples of microbial cells and/or suitable culture conditions for alcohol fermentation are disclosed in U.S. Patent Nos. 4310629, 7741 1 19, 7803623, 8679822, 7989206, 8247208, 8669076, 8093037, 9187743, 9441250 and 8394622, which are incorporated herein by reference.
  • compositions comprising: (i) a first lytic polysaccharide monooxygenase (LPMO) protein comprising an LPMO catalytic domain, wherein the first LPMO protein does not comprise a carbohydrate- binding module (CBM) (i.e., such an LPMO is a "truncated” or " ⁇ " LPMO), and (ii) a second LPMO protein comprising an LPMO catalytic domain, a linker, and a CBM (e.g., a "full-length" LPMO).
  • CBM carbohydrate- binding module
  • This type of composition can optionally be characterized as comprising at least a ACBM LPMO and a full-length LPMO.
  • such a composition can further comprise at least one cellulase. It is noted that, in some aspects, such a composition further having a cellulase can exhibit improved
  • a ACBM LPMO in certain aspects of a composition comprising a ACBM LPMO and a full-length LPMO can be any non-native LPMO as disclosed elsewhere herein.
  • a ACBM LPMO can lack a CBM, but comprise all or a certain minimal portion of a linker region, for example.
  • a ACBM LPMO can lack both CBM and entire linker regions as compared to a native counterpart LPMO.
  • an LPMO can optionally be prepared using a protease (e.g., papain) treatment approach.
  • a protease e.g., papain
  • a full-length LPMO can be expressed, followed by a suitable proteolytic treatment to remove the linker and CBM, thereby providing a ACBM LPMO.
  • the LPMO catalytic domain of a ACBM LPMO in certain aspects of a composition comprising a ACBM LPMO and a full-length LPMO can comprise an amino acid sequence that is 100% identical to, or at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, amino acid residues 22-248 of SEQ ID NO:2.
  • such a ACBM LPMO can comprise an amino acid sequence that is 100% identical to, or at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, amino acid residues 22-273 of SEQ ID NO: 10.
  • a full-length LPMO (i.e., second LPMO) of a composition comprising a ACBM LPMO and a full-length LPMO comprises an LPMO catalytic domain, a linker, and a
  • a full-length LPMO is a wild type, native form of an LPMO, such as any native LPMO disclosed elsewhere herein.
  • a "full-length" LPMO herein can be modified, such as by having an insertion, deletion, and/or amino acid substitution; however, such a modified "full-length" LPMO has a functional catalytic domain, linker, and CBM.
  • a full-length LPMO in some aspects can comprise an amino acid sequence that is 100% identical to, or at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to, amino acid residues 22-344 of SEQ ID NO:2.
  • a ACBM LPMO and a full-length LPMO comprised in a composition herein can be either autologous or heterologous with respect to each other.
  • LPMO proteins that are autologous can be derived from the same source protein (e.g., the ACBM LPMO can be derived from the corresponding full-length LPMO that natively comprises the amino acid sequence of the ACBM LPMO).
  • An example of such an autologous pair of LPMO proteins are amino acid residues 22-344 of SEQ ID NO:2 (mature HjGH61A) and amino acid residues 22-273 of SEQ ID NO: 10 (mature
  • LPMO proteins that are heterologous to each other are typically derived from LPMOs originating from different species. Pairs of heterologous LPMO proteins include, for example, amino acid residues 22-273 of SEQ ID NO: 10 (mature HjGH61 AwoCBM_2) with either of amino acid residues 20-336 of SEQ ID NO:24 (mature CtGH61 ) or amino acid residues 20-342 of SEQ ID NO:30 (mature StGH61 ), for example.
  • the ratio of a full-length LPMO to a ACBM LPMO in a composition herein can be about 50:50 in some aspects. Still, in some aspects, such a ratio can be, respectively, about 10:90, 20:80, 25:75, 30:70, 40:60, 50:50, 60:40, 70:30, 75:25, 80:20, or 90: 10.
  • a composition comprising a ACBM LPMO, a full-length LPMO, and at least one cellulase can exhibit improved saccharification activity on a cellulosic substrate compared to the saccharification activity of a composition that only differs from the former composition by lacking the ACBM LPMO.
  • Such an improvement can be by at least about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, for example.
  • the total protein amount of the combination of ACBM LPMO and full-length LPMO added to the composition is the same as the total protein amount of full-length LPMO added to the composition that lacks the ACBM LPMO.
  • a composition comprising a ACBM LPMO, a full-length LPMO, and at least one cellulase can be a cellulolytic enzyme composition (any as disclosed elsewhere herein) to which the ACBM LPMO and full-length LPMO proteins have been added. Such addition can be performed, for example, by directly adding these LPMO proteins to other enzymes in preparing a cellulolytic enzyme composition, or by expressing them in a cell that expresses all or most of the enzymes of the cellulolytic enzyme composition.
  • a cellulolytic enzyme composition can comprise enzymes of the Accellerase®- DUET cocktail, if desired. In some aspects, a cellulolytic enzyme composition
  • non-LPMO enzyme components in native form or that have been modified to remove their respective CBM's.
  • non-LPMO enzymes include cellobiohydrolases (e.g., CBH1 , CBH2), endoglucanases (e.g., EG1 , EG2), and endoxylanases (e.g., XynD).
  • cellobiohydrolases e.g., CBH1 , CBH2
  • endoglucanases e.g., EG1 , EG2
  • endoxylanases e.g., XynD
  • enzymes in modified form (- ⁇ ) include those comprising or consisting of amino acid residues 18-453 of SEQ ID NO:52
  • HjCEL7A-ACBM amino acid residues 25-389 of SEQ ID NO:56 (HjCEL6A-ACBM), amino acid residues 18-397 of SEQ ID NO:60 (HjCEL7B-ACBM), amino acid residues 22-348 of SEQ ID NO:62 (HjCEL5A-ACBM), or amino acid residues 20-336 of SEQ ID NO:66 (PfXynD-ACBM); corresponding native versions of these ACBM proteins that can be used are listed in Table 1 .
  • any of these native and/or ACBM non- LPMO saccharification enzyme components can comprise or consist of an amino acid sequence that is at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the foregoing amino acid sequences, and retain respective enzymatic activity. Any combination of, such as all of, these native and/or modified enzymes can be employed, for instance, in a cellulolytic enzyme composition herein.
  • a cellulolytic enzyme composition comprising a ACBM LPMO and full-length LPMO in some aspects can comprise one or more of a beta-glucosidase (e.g., from Magnaporthe grisea), beta- xylosidase (e.g., from Fusarium verticillioides, such as Fv3A or Fv43D), and/or an L- alpha-arabinofuranosidase (e.g., from Fusarium verticillioides, such as Fv51A); such enzyme(s) may optionally be in addition to one or more of the above native and/or ACBM non-LPMO enzymes.
  • a beta-glucosidase e.g., from Magnaporthe grisea
  • beta- xylosidase e.g., from Fusarium verticillioides, such as Fv3A or Fv43D
  • an L- alpha-arabinofuranosidase e
  • a composition comprising a ACBM LPMO, a full-length LPMO, and at least one cellulase in some embodiments can be used in a saccharification/hydrolysis reaction employing any feature (e.g., time, temperature, pH, amount and type of substrate) as disclosed elsewhere herein.
  • the initial percent dry solids (% w/v) of cellulose substrate (e.g., pretreated cellulose feedstock) in a hydrolysis reaction can be about, or at least about, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 15-20%, 15-25%, 15-18%, 15-20%, or 15-25%.
  • a hydrolysis reaction may comprise a cellulosic corn substrate such as one that has been pretreated (e.g., DACS).
  • a method for hydrolyzing a cellulosic substrate comprising contacting the substrate with a composition comprising a ACBM LPMO, a full-length LPMO, and at least one cellulase under aqueous conditions.
  • a composition comprising a ACBM LPMO, a full-length LPMO, and at least one cellulase under aqueous conditions.
  • compositions and methods disclosed herein include:
  • a non-native lytic polysaccharide monooxygenase (LPMO) protein comprising: an LPMO catalytic domain, and a linker or linker portion located C-terminal to the catalytic domain, wherein the linker or linker portion comprises an amino acid sequence that is (i) at least 12 residues in length and (ii) at least about 80% identical to an amino acid sequence located within the first 40 amino acid residues of a native LPMO linker or cellulase linker, wherein the non-native LPMO protein does not comprise a
  • CBM carbohydrate-binding module
  • linker or linker portion comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO:44 or SEQ ID NO:45.
  • AA9 auxiliary activity family 9 protein
  • a polynucleotide comprising a nucleotide sequence encoding a non-native LPMO protein according to any of embodiments 1 -5, optionally wherein one or more regulatory sequences are operably linked to the nucleotide sequence, and further optionally wherein the one or more regulatory sequences include a promoter sequence.
  • a cell comprising the polynucleotide of embodiment 6, optionally wherein the cell is capable of expressing the non-native LPMO protein encoded by the nucleotide sequence.
  • a method of producing a non-native lytic polysaccharide monooxygenase (LPMO) protein comprising: (a) providing a cell according to embodiment 7, wherein the cell is capable of expressing the non-native LPMO protein encoded by the nucleotide sequence; (b) incubating the cell under suitable conditions in which the cell expresses the non-native LPMO protein; and (c) optionally, isolating the non-native LPMO protein expressed in (b).
  • LPMO non-native lytic polysaccharide monooxygenase
  • a composition comprising a non-native LPMO protein according to any of embodiments 1 -5, and at least one cellulase, optionally wherein the composition is an enzyme cocktail for saccharifying cellulosic biomass.
  • a method for hydrolyzing a cellulosic substrate comprising contacting the substrate with a composition comprising a non-native LPMO protein according to any of embodiments 1 -5, and at least one cellulase, under aqueous conditions, and optionally producing alcohol by culturing a suitable microbial cell in a medium comprising glucose and/or glucose-containing oligosaccharide products of the hydrolysis.
  • a composition comprising: (i) a first lytic polysaccharide monooxygenase (LPMO) protein comprising an LPMO catalytic domain, wherein the first LPMO protein does not comprise a carbohydrate-binding module (CBM), and (ii) a second LPMO protein comprising an LPMO catalytic domain, a linker, and a CBM.
  • LPMO polysaccharide monooxygenase
  • composition of embodiment 12 further comprising at least one cellulase, optionally wherein the composition is an enzyme cocktail for saccharifying cellulosic biomass.
  • composition of embodiment 12 or 13 further comprising a cellulosic substrate, optionally wherein the composition comprises at least about 15% (w/v) of the substrate.
  • composition of embodiment 14, wherein the cellulosic substrate comprises at least one cellulosic corn substrate, optionally wherein the cellulosic corn substrate has been pretreated.
  • composition of embodiment 12, 13, 14, or 15, wherein the LPMO catalytic domain of the first LPMO comprises an amino acid sequence that is at least about 80% identical to amino acid residues 22-248 of SEQ ID NO:2,
  • the first LPMO comprises an amino acid sequence that is at least about 80% identical amino acid residues 22-273 of SEQ ID NO: 10.
  • a method for hydrolyzing a cellulosic substrate comprising contacting the substrate with a composition according to any of embodiments 12, 13, or 16, under aqueous conditions, and optionally producing alcohol by culturing a suitable microbial cell in a medium comprising glucose and/or glucose-containing oligosaccharide products of the hydrolysis.
  • H. jecorina GH61A full-length or truncated was diluted to a final concentration of 25 ⁇ in buffer (50 mM sodium acetate pH 5, or sodium phosphate pH 7).
  • the reference cell was supplemented with an equal volume of H. jecorina GH61A storage buffer (50 mM MES, pH 6, 100 mM NaCI).
  • the sample temperature was increased from 30 °C to 85 °C at a rate of 100 °C per hour or 60 °C per hour. After completion of the temperature ramp, samples were re-scanned to determine the reversibility of the unfolding transition.
  • H. jecorina GH61 A contains six tryptophans in its catalytic core. As such, folding transition can be monitored for both the full-length and truncated molecule using intrinsic fluorescence.
  • tryptophan fluorescence was measured across a temperature ramp from 30 °C to 85 °C.
  • the excitation and emission wavelengths were set to 280 nm and 332 nm, respectively, and the temperature was increased at a rate of 100 °C/hour or 60 °C/hour. Both full-length and truncated H.
  • jecorina GH61A were mixed to a final concentration of 10 ⁇ in either sodium acetate (50 mM pH 5) or sodium phosphate (50 mM pH 7) buffer.
  • Enzyme samples were added, based on mg total enzyme protein per g of glucan + xylan (DACS), or per g of glucan (PCS) ("mg/g” as used below refers to either of these dosage measures, depending on the substrate type) to triplicate vials containing 5 g of biomass slurry.
  • DAS glucan + xylan
  • PCS per g of glucan
  • Enzymes were diluted in 50 mM sodium acetate, pH 5.0, to obtain the desired loading concentrations. Vials were incubated at 50 °C, 200 rpm, for up to 72 hrs. At the end of the incubation period, the saccharification reaction was quenched with 100 mM glycine buffer pH 10. Quenched hydrolysate (200 ⁇ _) was transferred to a filter plate, and centrifuged at 3500 rpm for 5 m in to separate the supernatant from unreacted solids. Ten (10) ⁇ _ of the quenched supernatant was added to 200 ⁇ _ of MILLIQ water in a 96- well HPLC plate and the soluble sugars were measured by HPLC.
  • Phosphoric acid swollen cellulose (PASC) binding assays were conducted basically as previously described (Wood, 1988, In Methods of Enzymology, Vol. 160. Pp. 19-25. Academic Press, San Diego, CA). Purified GH61 enzymes (full-length and truncated) were serially diluted in triplicate 96-well microtiter plate wells (150 ⁇ _ each). In a separate tube, 6000 ⁇ _ of PASC (0.5% dry solids solution) was mixed with ascorbic acid (1 mM final concentration), 120 ⁇ _ copper sulfate (10 mM) and 4860 ⁇ _ buffer.
  • PASC Phosphoric acid swollen cellulose
  • the buffer was either 50 mM sodium acetate for pH 5, or 120 mM acetate/Bis-Tris buffer for pH 7.
  • the final PASC concentration was 0.25% dry solids. Plates were sealed with foil and incubated at 50 °C with shaking. PASC enzymatic solubilization was measured by a change in absorbance at 420 nm following incubation for 24-48 hours. PASC is a disordered form of cellulose. Avicel® Binding Assay
  • BioPolymer dry solids concentrations were incubated with 0.125 mg/mL GH61 protein.
  • Avicel® is a microcrystalline form of cellulose.
  • GH61 proteins were individually purified to a single band, as visualized by SDS- PAGE, in three steps: desalting with a column (HiTrapTM 5 mL), followed by
  • HIC hydrophobic interaction chromatography
  • SEC Size-exclusion chromatography
  • Trichoderma reesei strain QM6a genomic DNA and introduced via Gateway® cloning (Life Technologies) into the pTTTpyr2 vector to produce the pTTTpyr2-G/-/67/ ⁇ plasmid (pTTTpyr2 is similar to the pTTTpyrG vector described in PCT Appl. Publ. No.
  • SEQ ID NO: 1 The amino acid sequence encoded by SEQ ID NO: 1 is represented by SEQ ID NO:2; SEQ ID NO:3 represents the mature secreted form of SEQ ID NO:2.
  • FIGs. 1A-B depict the genomic DNA encoding full-length wild type H.
  • FIGs. 1A-B also show the positions of the primer pairs for preparing sequences encoding truncated GH61A proteins HjGH61AwoCBM_1 (SEQ ID NO:8) and HjGH61AwoCBM_2 (SEQ ID NO: 10).
  • the amino acid sequence of HjGH61AwoCBM_1 (SEQ ID NO:8) contains the signal sequence, catalytic domain, and the first ten residues of the linker (i.e., most of the linker, and the entire CBM, are deleted).
  • HjGH61AwoCBM_2 (SEQ ID NO: 10) contains the signal sequence, catalytic domain, and the first twenty-five residues of the linker (i.e., much of the linker, and the entire CBM, are deleted).
  • the mature, secreted forms of each of HjGH61 AwoCBM_1 (SEQ ID NO:8) and HjGH61AwoCBM_2 (SEQ ID NO: 10) were expected to lack the signal sequence (positions 1 -21 ) of each amino acid sequence, respectively.
  • HjGH61AwoCBM_1 or HjGH61AwoCBM_2 were individually cloned into the pTTTpyr2 vector with Gateway ® LR Clonase ® (Life Technologies).
  • Protoplasts of H. jecorina strain (Acbhi, cbhll, egl, egll, eglll, eglV, egV, egVI, man1, bgl1) were transformed with the individual pTTTpyr2_/-//G/-/67/4 or
  • pTTTpyr2_HjGH61 AwoCBM_1 or _2 vectors and grown on selective agar containing acetamide at 28 °C for 7 days as previously described in, for example, PCT Pat. Appl. Publ. No. WO2009/048488 (incorporated herein by reference).
  • 10 ⁇ _ of spore suspension was added to 200 ⁇ _ of a glycine minimal medium (PCT Pat. Appl. Publ. No. WO201 1/038019) supplemented with 2% glucose/sophorose mixture (U.S. Pat. No. 7713725).
  • the plates were incubated at 28 °C for 6 days with shaking at 220 rpm (INFORS incubator shaker). Protein samples were harvested by transferring the culture medium to a 96-well filter plate (Corning 3505) and collecting the filtrate under vacuum. Larger samples were prepared by fermentation in 1 -L reactors (Eppendorf D76FB04MBPD) and centrifugation to generate cell-free supernatants.
  • HjGH61AwoCBM_1 was only poorly expressed. As indicated above, HjGH61A lacking all of the linker and CBM domains was not expressed at any detectable level (data not shown). Western blot analyses confirmed these observations (data not shown).
  • the gBlocks® used for preparing certain full-length or C-terminally truncated GH61 coding sequences (7. saturni, S. thermophile, C. thermophilus) were amplified by PCR using the primers listed in Table 2.
  • the amplified coding sequences were first cloned into Gateway® pENTRTM (Life Technologies) according to the manufacturer's protocol. These constructs were then transformed into Invitrogen One Shot® TOP10 Competent Cells, grown overnight, isolated (Qiagen QIAprep® Spin Miniprep Kit), and sequenced (Sequetech, Mountain View, CA). Individual pENTRTM clones containing the respective correct sequence were then cloned by LR recombination (Gateway® Technology, Life Technologies) into the destination vector, pTTT/pyr2, according to the manufacturer's protocol. These constructs were also transformed into TOP 10 cells, grown overnight, isolated, and sequenced.
  • H. jecorina host strain (Acbhl, cbhll, egl, egll, eglll, eglV, egV, egVI, manl, xyn2, xyn3, bgll; PCT Pat. Appl. Publ. No. WO2010/141779) was transformed according to a modified version of the PEG-mediated fungal transformation protocol described by Penttila et al. (1987, Gene 61 : 155-164, incorporated herein by reference).
  • spores were grown for 16-24 h at 24 °C with shaking at 150 rpm in yeast extract growth (YEG) medium, which contained 5.0 g/L BD BACTO Yeast Extract and 22.0 g/L Glucose. H2O P. Germinating spores were harvested by
  • the transformation mixtures which contained about 1 g of DNA and 1 -5x10 7 protoplasts in a total volume of 200 ⁇ , were each treated with 2 mL of 25% PEG solution, diluted with 2 volumes of 1 .2 M sorbitol/10 mM Tris pH 7.5, 10 mM CaC , and mixed with 3% selective top agarose MM containing 5 mM uridine and 20 mM acetamide. The resulting mixtures were poured onto 2% selective agarose plates containing uridine and acetamide.
  • FIG. 3 also shows a longer conserved sequence that encompasses SEQ ID NO:44:
  • V/l-A-Q-G/S/R-S/T/K-S-A/V-A-T-A-T-A/G-S/T-A-T-P/L/V-P-G-G-G SEQ ID NO:45. This longer conserved sequence potentially plays a role in allowing ample expression of GH61 proteins.
  • thermophila GH61 LPMO
  • thermophila GH61 as its sequence is not as related to the above-tested GH61 proteins; e.g., whereas the catalytic domains (approx.) of HjGH61A, CtGH61 , StGH61 and TsGH61 have at least about 55-60% amino acid identity to each other (which supports broad applicability of the presently disclosed subject matter), the catalytic domain of MtGH61 only has about 32-37% amino acid identity with the foregoing catalytic domains. This observation indicates even broader applicability of the presently disclosed subject matter.
  • Table 4 indicates that both HjGH61A and HjGH61AwoCBM_2 demonstrate activity on PASC at both pH 5 and pH 7, in a 6-hour reaction at 50 °C. PASC activity is higher for both forms of the protein at pH 7.
  • FIGs. 10A-B provide the results of these analyses, indicating that the full-length wild type forms of the tested GH61 proteins exhibited greater Avicel® binding activity compared to their respective truncated counterparts lacking a CBM (and only having a partial linker).
  • DACS substrate was added to 5 g glass vials, with water and 6N sulfuric acid to achieve a slurry (5 g) of 15% (w/v) dry solids, pH 5.0. This preparation was placed at 50 °C with 200 rpm shaking, and allowed to incubate overnight. The following day, pH was measured and, if necessary, adjusted to 5.0-5.1 . Enzymes (DUET cocktail plus one of the GH61 's) were then added to achieve a constant 14 mg/g dose in which the HjGH61A or HjGH61AwoCBM_2 component ranged from 5%, 10%, 15%, 20% of the total dose (i.e., 0.7, 1.4, 2.1 , 2.8 mg/g).
  • reaction vials were incubated overnight at 50 °C with 200 rpm shaking. The pH of each reaction was again measured and adjusted (brought to pH 5.0-5.1 by adding 1 -3 ⁇ _ 6 N sulfuric acid), if necessary. The reactions were held for a total of 24-48 hours. After this incubation period, each vial was mixed thoroughly, taking care not to accumulate solids on the vial wall, and a wide-bore pipet was used to remove 500 ⁇ _ from the reaction. Each 500- ⁇ _ sample was transferred to an EPPENDORF tube and centrifuged at maximum velocity to pellet the solids.
  • HjGH61AwoCBM_2 addition (15% of total enzymes added) was chosen for subsequent experiments.
  • PCS dilute acid-pretreated corn stover
  • a dilute ammonia-pretreated switchgrass biomass preparation comprising 15% solids (which comprised 35% cellulose and 22% xylan) (pretreatment per U.S. Pat. Appl. Publ. No. 2007/0031918, which is incorporated herein by reference) and a hemicellulase cocktail (27% endo-xylanase, 47% beta-xylosidase, 25% L-alpha-arabinofuranosidase) was incubated for 48 hours (50 °C, pH 5.3, 200 rpm) to digest the xylan and make the cellulose more accessible. An acid-quenched, 0.2- ⁇ -filtered aliquot of the
  • hemicellulase-treated preparation was analyzed by HPLC, determining that 70% of the xylose and 8% of the glucose was released. The rest of the reaction was dewatered by filtering through cheesecloth, after which the solids were air-dried and milled to generate the substrate for further enzymatic hydrolysis.
  • GH61 proteins HjGH61 A, HjGH61AwoCBM_2, StGH61 , StGH61woCBM_2
  • a cellulase-containing enzyme cocktail to slurries (pH 7) containing 5%, 10%, or 15% solids of the above-prepared hemicellulase-treated switchgrass substrate to commence saccharification reactions.
  • Sugar monomer and oligomer products of each reaction were monitored by AMINEX-column chromatography following 1 or 2 days incubation.
  • HjGH61AwoCBM_2 were sometimes added at different times or in combination. These proteins were added at 15% of the total protein (14 mg/g) in saccharification reactions in which the added protein components were the DUET enzyme cocktail and one or both of HjGH61A or HjGH61AwoCBM_2.
  • the test conditions were as follows:
  • CBM-containing components CBH1 , CBH2, EG1 , EG2, HjGH61 A, PfXynD
  • a mixture was prepared in which 100% of these particular enzymes were the full-length versions or truncated (ACBM) versions; a mixture was also prepared in which there was a 50:50 combination of full-length and ACBM versions of each enzyme.
  • the rest of the enzymes in each of these three mixture types were Mg3A, Fv3A, Fv43D and Fv51A as listed in Table 7.
  • Hydrolysis with the three different enzyme mixtures was conducted against either 18% or 10% solids (DACS substrate), at pH 5.2, 50 °C and 200 rpm with a 12 mg/g total protein dose. Liquefaction of substrate was visually monitored and sugar release was measured by HPLC.
  • FIG. 1 1 shows the results of each saccharification reaction from 24-120 hours.
  • the reaction comprising the 50:50 combination of full-length and ACBM versions of enzymes resulted in faster liquefaction (data not shown) and faster hydrolysis (glucose yield, FIG. 1 1 ) than either the 100% full-length mixture or the 100% ACBM mixture.
  • This synergy was not similarly observed in reactions with 10% solids.
  • the 50:50 combination reaction had a glucose yield that was equivalent to the glucose yield of the 100% full-length reaction; both these reactions exceeded the rate and yield of the 100% ACBM reaction (FIG. 1 1 ).
  • Table 8 shows, for instance, that 50:50 combinations of each listed enzyme were used in saccharification reaction 15, while reaction 16 used only full-length versions of the listed enzymes. Saccharification reactions with one of each of the sixteen enzyme mixtures were conducted for 120 hours against 18% solids (DACS substrate), at pH 5.2, 50 °C and 200 rpm with a 12 mg/g total protein dose. Glucose, xylose and arabinose yields of each reaction are shown in the FIG. 12.
  • reaction 3 which had a 50:50 combination of full-length and truncated HjGH61A, but no other truncated enzyme type, yielded a glucose level higher than the glucose levels of those reactions having only full-length HjGH61A (reactions 1 , 2, 4, 5, 7, 14, 16).
  • reactions comprising other types of truncated enzymes only reached similarly high glucose yields when truncated HjGH61 A was also present; for example, compare reactions 6, 8, 10, 1 1 , 12, 13 and 15, which had certain truncated enzymes including truncated HjGH61A, with reactions 1 , 2, 4, 5, 7, 9 and 14, which had certain truncated enzymes but not truncated HjGH61A.
  • reaction 15, which had truncated versions of each enzyme present had a glucose yield similar to that of reaction 3 in which the only truncated enzyme was that of HjGH61A.
  • Different enzyme mixtures were prepared containing the ten enzymes (and amounts) as listed in Table 7 above (again, percent amounts of each component or combination thereof were of the 12 mg/g total protein dose).
  • Each mixture contained Mg3A, Fv3A, Fv43D and Fv51 A, and the full-length versions of each of CBH1 , CBH2, EG1 , EG2 and PfXynD.
  • the mixtures varied in terms of the ratio of full-length HjGH61 A to truncated (ACBM) HjGH61A. Saccharification reactions with 18% solids (DACS substrate) and one of each of the enzyme mixtures were conducted for 72 hours or 120 hours at pH 5.2, 50 °C and 200 rpm with a 12 mg/g total protein dose. Glucose, xylose and arabinose yields of these reaction are shown in the FIGs. 13A-B.
  • ACBM ACBM from H. jecorina, C. thermophilus and S. thermophile were used (details below).
  • Different enzyme mixtures were prepared containing most of the enzymes (and amounts) as listed in Table 7 above (percent amounts of each component were of a 9 mg/g total protein dose).
  • Each mixture contained Mg3A, Fv3A, Fv43D and Fv51A, and the full-length versions of each of CBH1 , CBH2, EG1 , EG2 and PfXynD.
  • the mixtures varied in terms of the HG61 component(s), which was either a single protein (StGH61 [full-length], StGH61woCBM_2 [ACBM], CtGH61 [full-length], CtGH61woCBM_2
  • Glucose, xylose and arabinose yields are shown in the FIG. 14.
  • the reaction comprising the 50:50 mix of full-length and truncated S. thermophile GH61 performed similarly to the reactions in which either of these proteins was used alone as the GH61 component.
  • the reaction comprising the 50:50 mix of full-length H. jecorina GH61A and truncated S. thermophile GH61 had marginally increased saccharification performance.
  • S. thermophile and/or H. jecorina GH61 proteins see first five column sets in FIG. 14
  • thermophile GH61 enzyme resulted in faster liquefaction (data not shown) and substantially increased hydrolysis (glucose yield).
  • C. thermophilus and/or H. jecorina GH61 proteins see the 6th to 8th column sets in FIG. 14
  • a substantial improvement of saccharification performance was only observed in the reaction with a 50:50 mix of truncated H. jecorina GH61 A and full-length C.
  • thermophilus GH61 enzyme thermophilus GH61 enzyme. These data altogether indicate that truncated HjGH61A can further enhance saccharification activity when combined with a full-length GH61 derived from a different organism, for example.

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Abstract

L'invention concerne des enzymes de polysaccharides mono-oxygénase lytiques (LPMO) tronqués, dépourvues d'un module de liaison aux glucides (CBM). Elle concerne également, par exemple, des compositions comprenant ces enzymes et des procédés de production et d'utilisation de celles-ci. Elle concerne en outre des combinaisons de LPMO tronqués et de LPMO pleine longueur pouvant présenter une activité améliorée de facilitation de la cellulase.
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US11297865B2 (en) 2019-08-16 2022-04-12 Cambridge Glycoscience Ltd Methods of treating biomass to produce oligosaccharides and related compositions
CN115895918A (zh) * 2022-10-14 2023-04-04 山东隆科特酶制剂有限公司 一种裂解性多糖单加氧酶及其应用
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