CN103189496B

CN103189496B - The fungal bacterial strain improved

Info

Publication number: CN103189496B
Application number: CN201180052763.5A
Authority: CN
Inventors: 蒂普纳斯·贝德亚洛伊; 伊什·库马尔·达万
Original assignee: Codexis Inc
Current assignee: Codexis Inc
Priority date: 2010-11-02
Filing date: 2011-11-01
Publication date: 2016-11-30
Anticipated expiration: 2031-11-01

Abstract

The invention provides the fungal bacterial strain of improvement.In some embodiments, the fungal bacterial strain of described improvement can be used for cellulosic material hydrolysis is become glucose.Present invention also offers by genetic modification to reduce by the fungal cell of amount of the endogenous cellobiose dehydrogenase activity of described emiocytosis, wherein said fungal cell is from cupreum section of section (Chaetomiaceae), and wherein said cell comprises the disappearance of cellobiose dehydrogenase 1 (cdh1) gene.

Description

The fungal bacterial strain improved

This application claims the U.S. Provisional Patent Application Serial No. 61/409,186,61/ all submitted on November 2nd, 2010 409,217, the U.S. Provisional Patent Application Serial No. that on June 16th, 61/409,472 and 61/409,480 and 2011 submits The priority of 61/497,661, passes through at this to quote to be incorporated by herein.

Invention field

The invention provides the fungal bacterial strain of improvement.In some embodiments, the fungal bacterial strain of described improvement can be used for Cellulosic material (cellulosic material) is hydrolyzed into glucose.

Background of invention

Cellulose is the polymer of simple sugars (simple sugar) glucose connected by β-1,4 glycosidic bond.Many micro- The enzyme of the glucosan of bio unboiled water solution β-connection.These enzymes include endoglucanase, cellobiohydrolase And β-glucosyl enzym (cellobiohydrolase).Endoglucanase is at random site digest cellulose polymer, by its dozen Open to be attacked by cellobiohydrolase.Cellobiohydrolase discharges cellobiose continuously from the end of cellulosic polymer Molecule.Cellobiose is the glucose dimer that water miscible β-1,4-connects.β-glucosyl enzym hydrolysis fiber disaccharide is Fructus Vitis viniferae Sugar.

Convert lignocellulosic material be ethanol have the advantage that big content of starting materials being readily available property, avoid burning or Needs and the total greenhouse gases reduced of land filling material produce.Wood, agricultural residue, herbaceous crops and municipal administration are solid Body waste material is contemplated by the raw material produced as ethanol.These materials are mainly made up of cellulose, hemicellulose and lignin. After cellulose is converted into glucose, glucose is easily ethanol by culture propagation.

Summary of the invention

The invention provides the fungal bacterial strain of improvement.In some embodiments, the fungal bacterial strain of described improvement can be used for Cellulosic material hydrolysis is become glucose.

The invention provides fungal cell, described fungal cell by genetic modification with reduce by described emiocytosis The amount of source property cellobiose dehydrogenase activity, wherein said fungal cell is from cupreum section of section (Chaetomiaceae), wherein Described cell comprises the disappearance of cellobiose dehydrogenase 1 (cdh1) gene.In some embodiments, described fungal cell is to ruin The species of the mould genus of silk (Myceliophthora).In some other embodiments, described fungal cell is thermophilic fungus destroyed wire (Myceliophthora thermophila).In some embodiments, described fungal cell by genetic modification with destroy The secreting signal peptide of cellobiose dehydrogenase.In some other embodiments, described fungal cell by genetic modification with Reduce the amount of the endogenous cellobiose dehydrogenase expressed by described cell.In some other embodiments, described fungus Cell by genetic modification with destroy encoding endogenous cellobiose dehydrogenase transcripton in translation initiation sequence.Again one In a little other embodiments, described fungal cell by genetic modification frameshift mutation to be incorporated into encoding endogenous fiber two In the transcripton of glucocorticoid dehydrogenase.In some other embodiments, described fungal cell by genetic modification to reduce coding The transcriptional level of the gene of endogenous cellobiose dehydrogenase.In some other embodiments, described fungal cell by Genetic modification is to destroy the promoter of the gene of encoding endogenous cellobiose dehydrogenase.In some embodiments, described very Bacterium cell by genetic modification to lack the gene of encoding endogenous cellobiose dehydrogenase at least in part.Other at some In embodiment, described fungal cell by genetic modification with reduce endogenous cellobiose dehydrogenase catalytic effect.One In a little other embodiments, the one or more residues in the avtive spot of the cellobiose dehydrogenase in described fungal cell By genetic modification.Again in some other embodiments, the haemachrome of the cellobiose dehydrogenase in described fungal cell One or more residues in binding structural domain are by genetic modification.

In some embodiments, the invention provides the fungal cell comprising cellobiose dehydrogenase.Implement at some In scheme, cellobiose dehydrogenase comprises and SEQ ID NO:2 at least about 85%, about 88%, about 90%, about 93%, about 95%, about 97%, about 98% or about 99% identical aminoacid sequence.In some other embodiments, fungal cell is modified to make Obtain described cell endogenous cellobiose of the amount that secretion reduces compared with the fungal cell before this modification or without this modification Dehydrogenase 1 (cdh1).

Present invention also offers and comprise two or more celluloses water-disintegrable enzyme (cellulose hydrolyzing Enzyme) enzymatic mixture, at least one in the wherein said water-disintegrable enzyme of two or more celluloses is thin by described fungus Cellular expression.In some embodiments, described enzymatic mixture is cell-free mixture.In some other embodiments, in advance The lignocellulose processed constitutes at least one substrate of this enzymatic mixture.In some other embodiments, pretreatment Lignocellulose comprises by selected from low-kappa number, ammonia pretreatment, steam explosion (steam explosion) and/or organic molten The lignocellulose of at least one disposal methods of agent extraction.

Present invention also offers the method for producing glucose, including making at least one cellulose matrix and comprising two kinds Or the enzymatic mixture contact of the more kinds of water-disintegrable enzyme of cellulose, in the wherein said water-disintegrable enzyme of two or more celluloses extremely Few a kind of fungal cell being provided by this article expresses.Present invention also offers the method for producing glucose, including making at least A kind of cellulose matrix contacts with at least one enzymatic mixture provided herein.In some other embodiments, described enzyme Mixture is cell-free mixture.In some other embodiments, described cellulose matrix is the wood fibre of pretreatment Element.Again in some other embodiments, the lignocellulose of pretreatment comprise by selected from low-kappa number, ammonia pretreatment, The lignocellulose of at least one disposal methods of steam explosion and/or organic solvent extraction.In the enforcement that some are other In scheme, the method for the present invention also includes glucose fermentation is become end-product.In some other embodiments, end-product is Fuel alcohol or precursor industrial chemical.In some other embodiments, described fuel alcohol is ethanol or butanol.Again at some In other embodiments, the method for the present invention, enzymatic mixture and/or fungal cell provide for this fungal cell be homology or At least one cellulose degrading enzyme of allos.

Present invention also offers and comprise as provided herein at least one fungal cell and/or at least one enzymatic mixture Fermentation medium.

Accompanying drawing is sketched

Fig. 1 provides the nucleotide sequence of thermophilic fungus destroyed wire (M.thermophila) CDH1 and aminoacid sequence (is respectively SEQ ID NO:1 and 2).

Fig. 2 provides the glucose measurement that shows to produce from 100g/kg glucosan (corn straw of pretreatment) The figure of the relative saccharifying effect of CF-200 with CF-400.At pH5, at 55 DEG C, reaction with 24.6% solid in 110 μ L volumes, and 128mM NaOAc, 3% enzyme are carried out.Use GOPOD to measure and measure glucose.Error bar represents ± 1SD, n=4.

Invention describes

The invention provides the fungal bacterial strain of improvement.In some embodiments, the fungal bacterial strain of described improvement can be used for Cellulosic material hydrolysis is become glucose.As noted herein, present invention provide for cellulose is changed into glucose The fungal bacterial strain improved.Especially, the fungal bacterial strain of improvement provided herein by genetic modification with reduce by described emiocytosis Endogenous cellobiose dehydrogenase activity amount.Before making the present invention, it is generally recognized that cellobiose dehydrogenase is by reducing fibre The concentration of dimension disaccharide enhances the speed of cellulose hydrolysis, and cellobiose is the potent inhibitor (ginseng of some fibre element enzyme component See such as Mansfield et al., Appl.Environ.Microbiol., 63:3804-3809 [1997]；With Igarishi etc. People, Eur.J.Biochem., 253:101-106 [1998]).Additionally, cellobiose dehydrogenase has been reported as dropping at cellulose Play a crucial role in terms of promoting collaborative enhancing by stoping hydrolyzate to suppress during solution (see, e.g. Hai et al., J.Appl.Glycosci.,49:9-17[2002]).Generally be additionally considered that cellobiose dehydrogenase lignocellulose is taken off wooden (delignifying) aspect is useful, thus the degraded of reinforcing fiber element.Recently, it has been reported that cellobiose dehydrogenase can increase Cellulolytic from glycosyl hydrolase family 61 strengthens albumen (cellulolytic enhancing protein) by force Activity (see for example, WO2010/080532A1) also can be used in order in the reaction of redox equilibrium purpose.

With the conventional understanding in this area differently, the invention provides the volume in the fungal cell producing cellulase The genetic modification (such as, disappearance) of the gene of code cellobiose dehydrogenase.This modification causes coming the cell of free genetic modification to divide The yield improvement of the fermentable sugars of the enzymatic mixture secreted.Therefore, the cellobiose of the organism secretion producing cellulase take off The minimizing of hydrogen enzyme provides the cellulase of the yield that can improve fermentable sugars during the enzyme hydrolysis of the substrate comprising cellulose Mixture.It addition, the disappearance of cdh gene (such as, encodes destination protein for introducing other sequences in fungal cell's genome Heterologous sequence) provide additional space.

Therefore, there is provided herein fungal cell, this fungal cell by genetic modification with reduce by described emiocytosis The amount of endogenous cellobiose dehydrogenase activity, wherein said fungal cell is from cupreum section of section, and wherein said fungus is thin Born of the same parents can secrete the enzymatic mixture comprising cellulase.In some embodiments, described fungal cell can secrete and comprise two Plant or the enzymatic mixture of more kinds of cellulase.In some embodiments, described fungal cell is the cupreum section of lower dependent of dead military hero Member: without hair Chaetomium (Achaetomium), atresia Thielavia (Aporothielavia), hair beak shell belong to (Chaetomidium), Chaetomium (Chaetomium), Corylomyces, rod softgel shell belong to (Corynascus), Fa Shi shell genus (Farrowia), Thielavia (Thielavia), handle spore shell belong to (Zopfiella) or the member of myceliophthora.Implement at some In scheme, the fungal cell of genetic modification provided herein is the member of the cupreum section selected from lower dependent of dead military hero: myceliophthora, shuttle spore Shell belongs to, rod softgel shell belongs to or Chaetomium.

Fungal taxonomy constantly experiences what arrangement was well recognized as.It is therefore contemplated that all aspects of the invention include by again The genus of classification and species, include but not limited to the organism of such as thermophilic fungus destroyed wire, and it has been given other title (examples multiple As, sporotrichum thermophile (Sporotrichum thermophile), sporotrichum thermophile (Sporotrichum Thermophilum), different fusarium globosum shuttle (Thielavia heterothallica), different rod softgel shell bacterium (Corynascus Heterothallica), thermophilic gold pityrosporion ovale (Chrysosporium thermophilum) and India to ruin silk mould (Myceliophthora indica)).It practice, expection the present invention include all epigamouss, phorozoon and synonym thereof, Basonym or taxonomy equivalent.

In some embodiments, fungal cell by genetic modification to reduce by the endogenous fiber of described emiocytosis The amount of disaccharide dehydrogenase activity.In some embodiments, described fungal cell is myceliophthora, Thielavia, Sporotrichum (Sporotrichum), rod softgel shell genus, Acremonium (Acremonium), Chaetomium, Ctenomyces (Ctenomyces), Scytalidium (Scytalidium), Talaromyces (Talaromyces) or thermophilic ascomycete belong to the species of (Thermoascus).Real at some Executing in scheme, described fungal cell is addicted to cellulose side spore mould (Sporotrichum cellulophilum), Tai Ruisisuo spore Shell bacterium (Thielavia terrestris), different rod softgel shell bacterium, different fusarium globosum shuttle, chaetomium globosum (Chaetomium Globosum), the basket bacterium of handle (Talaromyces stipitatus) or thermophilic fungus destroyed wire.In some embodiments, described Fungal cell is the fungal cell separated.

In some embodiments, described fungal cell by genetic modification to reduce by the endogenous of described emiocytosis The amount of cellobiose dehydrogenase.In some embodiments, described fungal cell is taken off to destroy cellobiose by genetic modification The secreting signal peptide of hydrogen enzyme.In some embodiments, described fungal cell by genetic modification with reduce by described cell table The amount of the endogenous cellobiose dehydrogenase reached.Such as, in some embodiments, described fungal cell by genetic modification with broken Bad translation initiation sequence, and in some other embodiments, described fungal cell by genetic modification so that frameshift mutation is introduced In the transcripton of encoding endogenous cellobiose dehydrogenase.In some other embodiments, described fungal cell is lost Pass the transcriptional level modifying the gene to reduce encoding endogenous cellobiose dehydrogenase.Such as, in some embodiments, institute State fungal cell by genetic modification to destroy the promoter of the gene of encoding endogenous cellobiose dehydrogenase.Such as, at some In embodiment, by using fungal cell described in the genetic modifications such as termination codon, terminator removal, transposon to destroy volume The gene of code endogenous cellobiose dehydrogenase.In some other embodiments, described fungal cell is by genetic modification For lacking the gene of encoding endogenous cellobiose dehydrogenase at least in part.In some other embodiments, described fungus Cell by genetic modification with reduce endogenous cellobiose dehydrogenase catalytic effect.In some embodiments, described very Bacterium cell has been so that the one or more residues in the avtive spot of cellobiose dehydrogenase are suddenlyd change by genetic modification.? In some embodiments, one or more residual in the haemachrome binding structural domain of the cellobiose dehydrogenase of described fungal cell Base is by genetic modification.It practice, be intended for modifying fungal cell to reduce the fiber expressed by described cell and/or secrete Any suitable means of the amount of two glucocorticoid dehydrogenases will can use in the present invention.

In some embodiments, in cellobiose dehydrogenase is included in EC1.1.99.18.In some embodiments In, described cellobiose dehydrogenase comprises and SEQ ID NO:2 at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical aminoacid Sequence.

In some embodiments, fungal cell also comprises at least one fiber encoded for this fungal cell is allos At least one gene of element digestive enzyme.Such as, in some embodiments, described fungal cell's process LAN encoding fiber element degraded The homology of enzyme such as β-glucosyl enzym or heterologous gene.In some embodiments, described fungal cell's process LAN β-glucoside Enzyme and by genetic modification with reduce by described emiocytosis endogenous cellobiose dehydrogenase activity amount.

Present invention also offers the enzymatic mixture comprising the water-disintegrable enzyme of two or more celluloses, wherein said two kinds or At least one of the water-disintegrable enzyme of more kinds of celluloses is expressed by fungal cell as provided herein.Such as, in some embodiments In, described fungal cell is active to reduce by the endogenous cellobiose dehydrogenase of described emiocytosis by genetic modification Amount cell, wherein said fungal cell is the member of lower dependent of dead military hero: myceliophthora, Thielavia, Sporotrichum, rod softgel shell belong to, Acremonium, Chaetomium, Ctenomyces, Scytalidium, Talaromyces or thermophilic ascomycete belong to.In some embodiments, described enzyme Mixture is cell-free mixture.In some other embodiments, the substrate of enzymatic mixture comprises the wooden fibre of pretreatment Dimension element.In some embodiments, the lignocellulose of pretreatment comprise by low-kappa number, ammonia pretreatment, steam explosion and/ Or the lignocellulose that organic solvent extraction processes.In some embodiments, enzymatic mixture also comprises for described fungus thin Born of the same parents are at least one cellulose degrading enzyme of allos.In some embodiments, the two or more kinds of cellulose are water-disintegrable At least one in enzyme is expressed by the fungal cell separated.

Present invention also offers the method for producing glucose, the method includes making the mixed of cellulose and at least two enzyme Compound contacts.Such as, in some embodiments, the method include making cellulose with comprise two or more celluloses hydrolysis Property enzyme enzymatic mixture contact, at least one of the wherein said water-disintegrable enzyme of two or more celluloses is by as described herein Fungal cell expresses.In some embodiments, the method include making cellulose with comprise two or more celluloses hydrolysis Property enzyme enzymatic mixture contact, at least one of the wherein said water-disintegrable enzyme of two or more celluloses is by by genetic modification To reduce the cell expression of the amount by the endogenous cellobiose dehydrogenase activity of described emiocytosis, wherein said fungal cell It is myceliophthora, Thielavia, Sporotrichum, rod softgel shell genus, Acremonium, Chaetomium, Ctenomyces, Scytalidium, Talaromyces Or thermophilic ascomycete belongs to.In some embodiments, these methods cause glucose and/or the fiber of the cellulose from hydrolysis The yield of disaccharide increases and reduces to the oxidation of the sugared product of oxidation from the cellobiose of the cellulose of hydrolysis, described oxidation Sugar product such as gluconolactone, gluconate (gluconate), gluconic acid (gluconic acid), cellobiose lactone And/or cellobionic acid.

In some embodiments, enzymatic mixture is cell-free mixture.In some other embodiments, cellulose Substrate comprises the lignocellulose of pretreatment.In some other embodiments, the lignocellulose of pretreatment comprises and passes through The wooden fibre of at least one disposal methods of such as low-kappa number, ammonia pretreatment, steam explosion and/or organic solvent extraction Dimension element.

In some embodiments, these methods also include glucose fermentation becomes end-product such as fuel alcohol or precursor work Industry chemicals.In some embodiments, fuel alcohol is ethanol or butanol.In some embodiments, these methods include making Cellulose with also comprise the enzymatic mixture to the cellulose degrading enzyme that described fungal cell is allos and contact.

The fungal cell that comprise any the embodiment above is also provided herein and/or comprises and derive from any one The fermentation medium of the enzymatic mixture of the fungal cell of the embodiment above.

Definition

Except as otherwise noted, lead to during otherwise the practice of the present invention includes molecular biology, protein engineering and microbiology Routine techniques in the range of the art technology often used.These technology are well-known and are described in people in the art In many textbooks known to Yuan and list of references works.Herein all patents mentioned above and below, patent application, Article and publication are incorporated by reference clearly at this herein.

Unless defined otherwise herein, all technology the most used herein and scientific terminology have as involved in the present invention The identical meanings that the those of ordinary skill in field is generally understood that.Many technology dictionaries are well known by persons skilled in the art.Although It is similar to or is equivalent to method described herein and any suitable method of material and material can be used in the practice of the invention, but It is to this document describes some preferred method and materials.Should be understood that and the invention is not restricted to described ad hoc approach, operating instruction And reagent, because these methods, operating instruction and reagent can use their situation to change according to those skilled in the art. Therefore, by being integrally more fully described, with reference to the application, the term the most and then defined.

It addition, unless context clearly indicates otherwise, otherwise as used herein odd number " (a) ", " one " and " should (the) " includes the referents of plural number (an).Numerical range includes the numeral defining this scope.Therefore, disclosed herein Each numerical range be intended to each narrower numerical range of including falling in the numerical range that this is wider, as these are narrower Numerical range all write on herein clearly as.It is also contemplated that the numeral of each maximum (or minima) disclosed herein Limit value includes the digital limit value of each lower (or higher), as the digital limit value of these lower (or higher) is all by clearly Write on herein as.Additionally, title provided herein be not to can by integrally with reference to the application and obtain this Bright various aspects or the restriction of embodiment.Therefore, the term the most and then defined is by integrally with reference to the application more Define fully.But, in order to contribute to understanding the present invention, some terms are defined below.Except as otherwise noted, otherwise distinguish Ground, nucleic acid is write from left to right with 5' to 3' direction；Aminoacid sequence is to write from left to right to carboxyl direction with amino.

As used herein, term " comprises " and synonym (that is, is equivalent to term " include with its inclusive implication (including) " and corresponding synonym) use.

As used herein, " substrate (the substrate) " effect by enzyme that refers to is converted or intends to be converted into another Plant material or the compound of compound.This term not only includes that single compound also includes the combination of compound, such as comprise to Few solution of a kind of substrate, mixture and other materials.

As used herein, " conversion " refers to that substrate is enzymatically converted to the product of correspondence." conversion percentages " refers to referring to Within a period of time, the percentage ratio of the substrate of product it is converted under fixed condition.It is therefoie, for example, cellobiose dehydrogenase (" CDH " Or " cdh ") " enzymatic activity " or " active " of polypeptide can be expressed as the substrate " conversion percentages " to product.

As used herein, " secretion activity " refers to exist in extracellular environment (extracellular environment) The enzymatic activity of the cellobiose oxidisability enzyme (cellobiose oxidizing enzyme) produced by fungal cell.Cell External environment it may be that such as, extracellular environment (extracellular milieu) such as culture medium.Secretion activity is secreted The total amount impact of cellobiose oxidisability enzyme, is also affected by the catalytic effect of the cellobiose oxidisability enzyme secreted.

As used herein, " minimizing of catalytic effect " refer to such as be provided by this article or this area it is also known that standard skill Art is measured, and cellobiose oxidisability enzyme is relative to the minimizing of the activity of the cellobiose oxidisability enzyme of unmodified.

As used herein, term " enzymatic mixture " refers to the combination of at least two enzyme.In some embodiments, at least two Plant enzyme to exist in the composition.In some other embodiments, enzymatic mixture is deposited in cell (such as, fungal cell) ?.In some embodiments, each of enzyme or some are by different fungal cells and/or different true present in enzymatic mixture Bacterium culture produces.In some further embodiments, present in enzymatic mixture, all enzymes are produced by same cell.? In some embodiments, enzymatic mixture comprises cellulase, and in some other embodiments, enzymatic mixture comprises fiber Enzyme beyond element enzyme.In some embodiments, enzymatic mixture comprises at least one cellulase and at least one cellulase Enzyme in addition.In some embodiments, enzymatic mixture comprises and includes but not limited to following enzyme: endo-xylanase (EC3.2.1.8), xylobiase (EC3.2.1.37), α-l-arabfuranglycosidase (EC3.2.1.55), α-glucose aldehyde Acid enzyme (EC3.2.1.139), acetyl group xylan esterase (EC3.1.1.72), Resina Ferulae acyl esterase (EC3.1.1.73), coumaric acyl Esterase (EC3.1.1.73), alpha-galactosidase (EC3.2.1.22), beta galactosidase (EC3.2.1.23), beta-mannase Enzyme (EC3.2.1.78), beta-Mannosidase (EC3.2.1.25), inscribe-polygalacturonase (EC3.2.1.15), pectin Methyl esterase (EC3.1.1.11), inscribe-Galactanase (EC3.2.1.89), pectin acetyl base esterase (EC3.1.1.6), interior Cut-pectin lyase (EC4.2.2.10), transelminase (EC4.2.2.2), α rhamnosidase (EC3.2.1.40), outer Cut-galacturonic acid enzyme (EC3.2.1.82), circumscribed-galacturonic acid enzyme (EC3.2.1.67), circumscribed polygalacturonic acid split Solve enzyme (EC4.2.2.9), rhamnosan galacturonic acid inscribe lyases (rhamnogalacturonan endolyase) EC (4.2.2.B3), rhamnosan galacturonic acid acetylesterase (EC3.2.1.B11), rhamnosan galacturonic acid galacturonic acid Acid hydrolysis enzyme (EC3.2.1.B11), inscribe-arabanase (EC3.2.1.99), laccase (EC1.10.3.2), manganese rely on Property peroxidase (EC1.10.3.2), amylase (EC3.2.1.1), glucoamylase (EC3.2.1.3), lipase, lignin Peroxidase (EC1.11.1.14) and/or protease.

In some other embodiments, the present invention also provide for including at least one extension albumen (expansin) and/ Or extension protein like proteins, such as the filament expansion factor (swollenin) (see for example, Salheimo etc., Eur.J.Biochem., 269:4202-4211 [2002]) and/or filament expansion because of the enzymatic mixture of increment albumen.Extension albumen With cell wall structure lax relevant during plant cell growth.Have pointed out extension albumen and destroy cellulose and other cell wall Hydrogen bond between polysaccharide and there is no hydrolysing activity.In this way, it is believed that they allow slip and the cell wall of cellulose fibre Extension.The filament expansion factor is a kind of extension protein like proteins, including N end carbohydrate binding module family 1 domain (CBD) and C end extension protein-like structural domain.In some embodiments, extension protein like proteins and/or filament expansion are because of increment Albumen include in this type of domain one or two and/or destroy cell wall structure (such as destroying cellulosic structure), appoint Selection of land does not produce the reducing sugar of detectable amount.In some other embodiments, enzymatic mixture includes that cellulose integrates egg At least one polypeptide of (cellulose integrating protein), scaffolding protein and/or scaffolding protein sample albumen produces in vain Thing is (such as respectively from Clostridium thermocellum (Clostridium thermocellum) or solution fiber clostridium (Clostridium Cellulolyticum) CipA or CipC).In some other embodiments, enzymatic mixture includes at least one fiber Element induced protein and/or cellulose regulation albumen (cellulose induced protein and/or modulating Protein) (such as, by from cip1 or the cip2 gene of trichoderma reesei and/or the albumen of similar coded by said gene；See example As, Foreman etc., J.Biol.Chem., 278:31988-31997 [2003]).In some other embodiments, enzyme mixes If at least one member that compound each of includes at least one GH61 and aforementioned polypeptides classification, a polypeptide classification Dry member or any combination of these polypeptide classifications, to provide the enzymatic mixture being suitable to multiple use.At least one, two kinds, three Kind, four kinds, five kinds or can be used in multiple enzymatic mixture provided herein more than any combination of five kinds of enzymes and/or polypeptide.Thing In reality, do not expect that the enzymatic mixture of the present invention is limited to any specific enzyme, polypeptide, combination, because any applicable enzymatic mixture Can be used for the present invention.

As used herein, term " sugared " refers to any carbohydrate, including monosaccharide (such as, glucose, ribose, fructose, Galactose etc.), disaccharide (such as, sucrose, lactose, maltose, cellobiose, trehalose, 6-(.alpha.-D-galactosido)-D-glucose. etc.), oligosaccharide (such as, cotton seed Sugar, stachyose, amylose etc.) and polysaccharide (such as, starch, glycogen, cellulose, chitin, xylan, Arabinoxylo gather Sugar, mannan, fucoidan, galactomannan, callose, laminarin, chrysolaminarin, side chain form sediment Powder, glucosan, dextrin, maltodextrin, inulin, oligofructose, polydextrose etc.).Simple carbon hydrate contained in this term Thing and compound carbohydrate.It is true that do not expect that the present invention is limited to any specific sugar, because multiple sugar and sugar Form can be used for the present invention.

As used herein, term " sugared water-disintegrable enzyme (saccharide hydrolyzing enzyme) " is to hydrolyze at least A kind of any enzyme of sugar.

As used herein, term " cellobiose oxidisability enzyme " refers to the enzyme of oxidized fibre disaccharide.In some embodiments In, cellobiose oxidisability enzyme includes cellobiose dehydrogenase (EC1.1.99.18).

As used herein, term " cellobiose dehydrogenase ", " CDH " and " cdh " refers to cellobiose: acceptor (acceptor) 1-oxidoreductase, this enzyme catalysis fibre disaccharide in the presence of acceptor is converted into cellobiose-1, in 5- Ester and the acceptor of reduction.The example of cellobiose dehydrogenase falls in enzyme classification (E.C.1.1.99.18).Generally 2,6-dichloro Indophenols may act as acceptor, ferrum especially Fe (SCN)₃, molecular oxygen, ubiquinone or cytochrome C and other Polyphenols (polyphenolic) such as lignin also acts as acceptor.The substrate of this enzyme includes cellobiose, fiber-oligosaccharide, breast Sugar and D-glucityl-1,4-β-D-MANNOSE, glucose, maltose, mannobiose, sulfur are for cellobiose, galactosyl-manna Sugar, 1,4-.beta.-Xylobiose, xylose.Although electron donor includes that glucose or mannose are positioned at β-Isosorbide-5-Nitrae-two hexasaccharide of reduction end, but It is that α-1,4-hexoside, hexose, pentose and β-1,4 pentose polymer (pentomer) may act as at least some enzyme in these enzymes Substrate (see, e.g. Henriksson et al., Biochim.Biophys.Acta Prot.Struct.Mol.Enzymol., 1383:48-54[1998]；With Schou et al., Biochem.J., 330:565-571 [1998]).In some embodiments, Purpose cellobiose dehydrogenase in the present invention is by the CDH1 of cdh1 gene code.

As used herein, term " aoxidizes ", " oxidation () " and similar terms used herein refer to that one or more are fine The enzymatic of dimension disaccharide oxidation product is formed.When mentioning that the percentage ratio of cellobiose of oxidation uses, these percentage ratio reflection phase Percentage by weight (w/w) for the primary quantity of substrate.Such as, when enzymatic mixture contacts with cellobiose, the fiber of oxidation The percentage ratio reflection of disaccharide is relative to the percentage by weight (w/w) of the primary quantity of cellobiose present in solution.When enzyme mixes When thing contacts with cellulose matrix, the percentage ratio reflection of the cellobiose of oxidation is based on producing from the cellulose of total hydrolysis The percentage by weight (w/w) (wt%) of maximum (that is, Gmax) of glucose.

As used herein, " cellulose " refers to the polymer of the simple sugar glucose connected by β-Isosorbide-5-Nitrae glycosidic bond.

As used herein, " cellobiose " refers to the glucose dimer of water miscible β-Isosorbide-5-Nitrae-connection.

As used herein, term " cellodextrin " refers to that the glucose polymer of different length (that is, comprises at least two Portugal Grape sugar monomer).Each glucose monomer is connected by β-1,4 glycosidic bond.Cellodextrin is classified by its degree of polymerization (DP), polymerization The number of the glucose monomer that degree this cellodextrin of expression comprises.Modal cellodextrin is: cellobiose (DP=2)；Fiber Trisaccharide (DP=3)；Cellotetrose (DP=4)；Cellopentaose (DP=5)；And cellohexose (DP=6).In some embodiments, Cellodextrin has the DP (that is, cellobiose, cellotriose, cellotetrose, cellopentaose and/or cellohexose) of 2-6.One In a little embodiments, cellodextrin has the DP more than 6.The degree of polymerization that can measure cellodextrin molecule (such as by mass spectrum, is wrapped Include but be not limited to substance assistant laser desorpted/ionization (MALDI) mass spectrum and electron spray ionisation ion trap (ESI-IT) mass spectrum).Survey The method of the degree of polymerization of amount cellodextrin molecule be known in the art (see, e.g., Melander et al., Biomacromol.,7:1410-1421[2006])。

As used herein, term " cellulase " refers to any enzyme of degraded cellulose.Therefore, energy contained in this term Enough hydrocelluloses (beta-1,4-glucan or β-D-glucoside bond) are shorter cellulose chain, oligosaccharide, cellobiose and/or Portugal The enzyme of grape sugar." cellulase " is divided into the enzyme of three subclass: 1,4-callose glucan hydrolase (1,4-β-D- Glucan glucanohydrolase) (" endoglucanase " or " EG ")；1,4-callose cellobiohydrolase (" exoglucanase ", " cellobiohydrolase " or " CBH ")；With β-D-glucoside-glucoside hydrolytic enzyme (" β-glucoside Enzyme ", " cellobiase ", " BG " or " BGL ").These enzymes act on being catalyzed the hydrolysis of the substrate of cellulose jointly.Inscribe Portugal Dextranase fracture interior keys also destroys the crystal structure of cellulose, exposes an other cellulose polysaccharide chain (" glucosan ").Fiber Disaccharide-hydrolysing enzymes progressively shortens dextran molecule, and main release cellobiose unit be (water-soluble beta-Isosorbide-5-Nitrae-connection of glucose Dimer) and glucose, cellotriose and cellotetrose.Cellobiose is split into monomer by β-glucosyl enzym.Cellulase Generally including synergism to destroy cellulose is solvable disaccharide or the different types of cellulose of oligosaccharide such as cellobiose The mixture of decomposability enzyme (cellulolytic enzyme) (endoglucanase and cellobiohydrolase), described disaccharide Or oligosaccharide is hydrolyzed to glucose subsequently further by β-glucosyl enzym.Cellulase is produced by multiple-microorganism.From thread very The cellulase (and hemicellulase) of bacterium and some antibacterials is widely used in many commercial Application, including processing natural fiber For saccharide.

As used herein, " fungal cell of cellulase-producing " is to produce at least one cellulase (that is, " cellulose water Solve property enzyme ") fungal cell.In some embodiments, the fungal cell of cellulase-producing provided herein expresses and secretion The mixture of the water-disintegrable enzyme of cellulose.

As used herein, term " the water-disintegrable enzyme of cellulose ", " cellulolytic enzyme " and similar terms refer to destroying Cellulose is the enzyme worked during solvable disaccharide or oligosaccharide such as cellobiose, described disaccharide or oligosaccharide subsequently by β- Glucosidase is hydrolyzed to glucose further.The mixture of the water-disintegrable enzyme of cellulose is also known as " cellulase ", " containing fibre herein The mixture of dimension element enzyme " and/or " cellulase mixture ".

As used herein, term " endoglucanase " and " EG " refer to the inside β-Isosorbide-5-Nitrae glucosides of a class catalysis fibre element The cellulase (EC3.2.1.4) of key hydrolysis.Term " endoglucanase " be further defined as herein inscribe-1,4-(1, 3;Isosorbide-5-Nitrae)-callose 4-glucan hydrolase (E.C.3.2.1.4), its catalysis fibre element, cellulose derivative (such as carboxylic Methylcellulose and hydroxyethyl cellulose), Isosorbide-5-Nitrae-β-D-glycosidic bond in lichenin, β-1 of mixing, 3 glucosans such as paddy Class callose or xyloglucan and the endo hydrolysis of β-1,4 key in comprising the other plant material of cellulosic component. The increase of the reducing end under neutral that endoglucanase activity based on matrix viscosity can reduce or be determined by reducing sugar inspection comes really Fixed (see for example, Zhang et al., Biotechnol.Adv., 24:452-481 [2006]).For the purposes of the present invention, inscribe Dextranase activity use carboxymethyl cellulose (CMC) hydrolysis determine (see for example, Ghose, Pur.Appl.Chem., 59: 257-268[1987])。

As used herein, " EG1 " refers to that be sorted under EC3.2.1.4 is catalyzed from encoding glycosyl hydrolase (GH) family 7 The carbohydrate activity enzyme that the nucleotide sequence of domain is expressed, or its any albumen, polypeptide or catalysis active fragment.At some In embodiment, EG1 is functionally connected in carbohydrate binding module (CBM), such as family 1 cellulose binding domain.

As used herein, term " EG2 " refer to be sorted under EC3.2.1.4 from encoding glycosyl hydrolase (GH) family 5 The carbohydrate activity enzyme that the nucleotide sequence of catalyst structure domain is expressed, or its any albumen, polypeptide or catalysis active fragment.? In some embodiments, EG2 is functionally connected in carbohydrate binding module (CBM), such as family 1 cellulose binding Territory.

As used herein, term " EG3 " refer to be sorted under EC3.2.1.4 from encoding glycosyl hydrolase (GH) family 12 The carbohydrate activity enzyme that the nucleotide sequence of catalyst structure domain is expressed, or its any albumen, polypeptide or catalysis active fragment.? In some embodiments, EG3 is functionally connected in carbohydrate binding module (CBM), such as family 1 cellulose binding Territory.

As used herein, term " EG4 " refers to from encoding glycosyl hydrolase (GH) family 61 being sorted in EC3.2.1.4 The carbohydrate activity enzyme that the nucleotide sequence of catalyst structure domain is expressed, or its any albumen, polypeptide or catalysis active fragment.? In some embodiments, EG4 is functionally connected in carbohydrate binding module (CBM), such as family 1 cellulose binding Territory.

As used herein, term " EG5 " refer to be sorted under EC3.2.1.4 from encoding glycosyl hydrolase (GH) family 45 The carbohydrate activity enzyme that the nucleotide sequence of catalyst structure domain is expressed, or its any albumen, polypeptide or catalysis active fragment.? In some embodiments, EG5 is functionally connected in carbohydrate binding module (CBM), such as family 1 cellulose binding Territory.

As used herein, term " EG6 " refer to be sorted under EC3.2.1.4 from encoding glycosyl hydrolase (GH) family 6 The carbohydrate activity enzyme that the nucleotide sequence of catalyst structure domain is expressed, or its any albumen, polypeptide or catalysis active fragment.? In some embodiments, EG6 is functionally connected in carbohydrate binding module (CBM), such as family 1 cellulose binding Territory.

As used herein, term " cellobiohydrolase " and " CBH " refer to a class of the glycosidic bond in hydrocellulose Cellulase (EC3.2.1.91).Term " cellobiohydrolase " is further defined as 1,4-callose fiber herein Disaccharide-hydrolysing enzymes (E.C.3.2.1.91), its catalysis fibre element, cell-oligosaccharide or comprise the glucose of any β-Isosorbide-5-Nitrae-connection Isosorbide-5-Nitrae-β-D-glucoside bond hydrolysis in polymer, (sees example from reproducibility or the non reducing end release cellobiose of chain As, Teeri, Tr.Biotechnol., 15:160-167 [1997]；With Teeri et al., Biochem.Soc.Trans., 26: 173-178[1998]).In some embodiments, cellobiohydrolase activity uses fluorescence two sugar derivatives 4-methyl umbrella Shape ketone group-. β .-D-lactoside determines and (see for example, van Tilbeurgh et al., FEBS Lett., 149:152-156 [1982]；With van Tilbeurgh and Claeyssens, FEBS Lett., 187:283-288 [1985]).

As used herein, term " CBH1 " and " 1 fiber type disaccharide-hydrolysing enzymes " refer to be sorted under EC3.2.1.91 from The carbohydrate activity enzyme that the nucleotide sequence of encoding glycosyl hydrolase (GH) family 7 catalyst structure domain is expressed, or its any egg In vain, polypeptide or catalysis active fragment.In some embodiments, CBH1 is functionally connected in carbohydrate binding module (CBM), such as family 1 cellulose binding domain.

As used herein, term " CBH2 " and " 2 fiber type disaccharide-hydrolysing enzymes " be the finger that is sorted under EC3.2.1.91 from The carbohydrate activity enzyme that the nucleotide sequence of encoding glycosyl hydrolase (GH) family 6 catalyst structure domain is expressed, or its any egg In vain, polypeptide or catalysis active fragment.2 fiber type disaccharide-hydrolysing enzymes are also usually referred to as " Cel6 family ".In some embodiments, CBH2 is functionally connected in carbohydrate binding module (CBM), such as family 1 cellulose binding domain.

As used herein, term " β-glucosyl enzym ", " cellobiase " and " BGL " refer to that catalysis fibre disaccharide is hydrolyzed to One fibrid element enzyme (EC3.2.1.21) of glucose.Term " β-glucosyl enzym " is further defined as β-D-glucoside herein Enzyme glucohydralase (E.C.3.2.1.21), the hydrolysis of its catalysis end irreducibility β-D-Glucose residue, with β-D-Portugal The release of grape sugar.Beta-glucosidase activity can use any suitable method to determine and (see for example, J.Basic Microbiol.,42:55-66[2002]).The beta-glucosidase activity of 1 unit is defined as at 40 DEG C, pH5, containing 0.01%In the 100mM sodium citrate of 20, from the 1mM p-nitrophenyl-β-D-pyranglucoside every point as substrate Clock produces 1.0pmol paranitrophenol.

As used herein, term " glycoside hydrolase 61 " and " GH61 " refer to the cellulase connection other with one or more Close a fibrid element enzyme of reinforcing fiber element hydrolysis when using.The GH61 family of cellulase is described in such as, carbohydrate Organized enzyme (CAZY) data base (see for example, Harris et al., Biochem., 49 (15): 3305-16 [2010]).

" hemicellulase " refers to the polysaccharide such as oligosaccharide that hydrolysis of hemicellulose is become little by catalysis as used herein Or the polypeptide of monomer sugar.Hemicellulose includes xylan, glucuronoxylan, araboxylan, glucomannan and wood Glucosan.Hemicellulase includes such as, below: endo-xylanase, xylobiase, α-l-arabfuranglycosidase, α-D-glycuronidase, Resina Ferulae acyl esterase, coumaric acyl esterase, alpha-galactosidase, beta galactosidase, 'beta '-mannase and Beta-Mannosidase.

As used herein, term " xylanolytic activities " and " activity (xylanolytic of decomposition xylan Activity) " at the biological activity being defined herein as the hydrolysis material containing xylan.Measure the activity decomposing xylan two kinds Basic skills includes: (1) measures total activity decomposing xylan, and (2) measure the activity (inscribe individually decomposing xylan Xylanase, xylobiase, arabinofuranosidase, α-glycuronidase, acetyl group xylan esterase, Resina Ferulae acyl esterase With α-glucuronyl-esterase) (see for example, Biely and Puchard, J.Sci.Food Agr.86:1636-1647 [2006]；Spanikova and Biely, FEBS Lett., 580:4597-4601 [2006]；With Herrmann et al., Biochem.J.,321:375-381[1997])。

Total xylanolytic activities can be determined by from polytype xylan, including Herba bromi japonici, emmer wheat, mountain hair Reducing sugar that Zelkova schneideriana Hand.-Mazz. and Larch polysaccharide are formed or determine the dyeing that the xylan dyeed from multiple covalency discharges by luminosity Xylan fragments measure.Common total activity assay decomposing xylan is based on from polymerization 4-O-methylglucuronic acid Xylan produces reducing sugar (see for example, Bailey et al., J.Biotechnol., 23:257-270 [1992]).Real at some Executing in scheme, xylanolytic activities is by measuring beech wood xylan (Sigma Chemical under following representative condition Co., Inc., St.Louis, Mo., USA) determined by the increase of xylan-digestive enzyme hydrolysis: 1mL reaction, 5mg/mL substrate (total solid), 5mg decompose the albumen/g substrate of xylan, 50mM sodium acetate pH 5,50 DEG C, 24 hours, use para hydroxybenzene first The glycan analysis (see for example, Lever, Anal.Biochem., 47:273-279 [1972]) that hydrazides (PHBAH) is checked.

As used herein, term " xylanase activity " refers to be catalyzed the inscribe of the Isosorbide-5-Nitrae-β-D-xylose glycosidic bond in xylan 1,4-β-D-xylan-xylan hydrolysis the enzymatic activity (E.C.3.2.1.8) of hydrolysis.In some embodiments, xylanase Activity uses beech wood xylan to determine as substrate.The xylanase activity of 1 unit is defined as at 50 DEG C, and pH5 is containing 0.01%In the 50mM sodium acetate of 20, rise beech wood xylan from the 2g/ as substrate initial hydrolyze In stage, generation 1.0 μm ol reducing sugar per minute (is measured with glucose equivalent；See for example, Lever, Anal.Biochem., 47:273-279[1972])。

As used herein, term " xylobiase activity " refers to be catalyzed the circumscribed-water of short β (1 → 4)-oligomeric xylose Solution to remove the β-D-xyloside xylan hydrolysis enzyme (E.C.3.2.1.37) of D-xylose residues one by one from non reducing end. In some embodiments of the present invention, the xylobiase activity of 1 unit is defined as at 40 DEG C, pH5, containing 0.01%In the 100mM sodium citrate of 20, from the 1mM p-nitrophenyl-β-D-xyloside generation per minute as substrate 1.0 μm ol paranitrophenols.

As used herein, term " acetyl group xylan esterase activity " refers to be catalyzed acetyl group from the xylan of polymerization, second Acylated xylose, acetylizad glucose, Alpha-Naphthyl acetas and the Carboxylesterase Activity of p-nitrophenyl Acetate hydrolysis (EC3.1.1.72).In some embodiments of the present invention, acetyl group xylan esterase activity is comprising 0.01%The 50mM sodium acetate pH5.0 of 20 use 0.5mM p-nitrophenyl yl acetate determine as substrate.1 unit Acetyl group xylan esterase activity be defined as at pH5,25 DEG C, it is possible to release 1pmol paranitrophenol anion per minute The amount of enzyme.

As used herein, term " Resina Ferulae acyl esterase active " refers to be catalyzed 4-hydroxy-3-methoxy cinnamoyl (Resina Ferulae acyl) Base, from the sugar of esterification, is typically the arabinose in " naturally " substrate, and hydrolysis is to produce ferulic acid (4-hydroxy-3-methoxy meat Cinnamic acid) 4-hydroxy-3-methoxy cinnamoyl-glycosylhydrolase activity (EC3.1.1.73).Resina Ferulae acyl esterase is also known as Resina Ferulae Acid esters enzyme, hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I or FAE-II.At this In some bright embodiments, Resina Ferulae acyl esterase active uses 0.5mM p-nitrophenyl ferulic acid in 50mM sodium acetate pH5.0 Ester determines as substrate.The Resina Ferulae acyl esterase active of 1 unit is equal at pH5,25 DEG C, it is possible to release 1 μm ol per minute is to nitre The amount of the enzyme of base phenol anion.

As used herein, term " α-glucuronic acid enzymatic activity " refers to that being catalyzed α-D-glucuronic acid glycosides is hydrolyzed to D-glucose aldehyde Acid and the α-D-glucosiduronic acid glucuronic acid hydrolytic enzyme activities (EC3.2.1.139) of alcohol.The α of 1 unit-glucuronic acid enzymatic activity etc. In at pH5,40 DEG C, it is possible to the amount of the enzyme of release 1pmol glucuronic acid per minute or 4-O-methylglucuronic acid (see for example, de Vries,J.Bacteriol.,180:243-249[1998])。

As used herein, term " α-l-arabfuranglycosidase activity " refers to the end being catalyzed in α-L-arabinose glycosides α-L-arabinofuranosidase glucosides arabinofuranosyl the hydrolytic enzyme of the hydrolysis of end irreducibility α-L-arabinofuranosidase glucosides residue (alpha-L-arabinofuranoside arabinofuranohydrolase) activity (EC3.2.1.55).This enzymatic activity is made For α-L-arabinofuranosidase glucosides, the α-L-arabinan (alpha-L-that comprises (1,3)-key and/or (1,5)-key Arabinan), araboxylan and arabinogalactan.α-l-arabfuranglycosidase is also known as galactoside Enzyme, α-arabinosidase, α-L-arabinose glycosides enzyme, α-arabinofuranosidase, arabinofuranosidase, polysaccharide α- L-arabinofuranosidase, α-L-arabinofuranosidase glucosides hydrolytic enzyme, L-arabinose glycosides enzyme and α-L-arabanase. For the purposes of the present invention, α-l-arabfuranglycosidase activity is identified below: use the 5mg of every mL100mM sodium acetate pH5 Moderately viscous Semen Tritici aestivi arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co.Wicklow, Ireland) continue 30 minutes at 40 DEG C with cumulative volume 200 μ L, pass through subsequentlyHPX-87H The arabinose analysis of column chromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Enzymatic lignin depolymerization can be by the lignin peroxidase of usual co-action, manganese peroxidase, lacca Enzyme and cellobiose dehydrogenase (CDH) complete.These exoenzymes commonly referred to as " lignin modification enzyme that lignin degradation is required (lignin-modifying enzyme) " or " LME ".In these enzymes three kinds include two kinds of glycosylated mistakes containing haemachrome Oxide enzyme: lignin peroxidase (LIP)；Mn dependency peroxidase (MNP)；Phenol oxidase with cupric: lacca Enzyme (LCC).Although the details of the reaction equation of lignin biodegradation understands the most completely, without being bound by theory, imply this A little enzymes utilize free radical for depolymerization reaction.

As used herein, term " laccase " refers to the oxidation of the cupric found in many plants, fungus and microorganism Enzyme.Laccase has enzymatic activity and carries out the oxidation of an electronics phenols with similar molecule.Laccase can be polymerization And enzymatic activity form can be dimer or trimer.

As used herein, term " Mn dependency peroxidase " refers to need the peroxidase of Mn.Mn dependency mistake The enzymatic activity of oxide enzyme (MnP) depends on Mn²⁺.Without being bound by theory, it has been suggested that the Main Function of this enzyme is by Mn²⁺Oxygen Chemical conversion Mn³⁺(see for example, Glenn etc., Arch.Biochem.Biophys., 251:688-696 [1986]).Subsequently, at the bottom of phenols The Mn3+ oxidation that thing is generated.

As used herein, term " lignin peroxidase " refers to the oxidation of the weak solution of catalysed in vitro polymeric lignin The outer haemachrome of the born of the same parents of depolymerisation.The substrate of some LiP, most notably 3,4-dimethoxy-benzyl alcohol (veratryl alcohol, VA), It it is the active redox compound showing and serving as redox mediator (mediator).VA is simultaneously by yellow archespore hair with LiP The lignin of flat lead fungi (P.chrysosporium) decomposes the secondary metabolite that culture produces, and without being bound by theory, Propose VA as lignin internal LiP be catalyzed oxidation in physiology's redox mediator work (see for example, Harvey, etc., FEBS Lett.195:242-246 [1986]).

As used herein, term " glucoamylase " (EC3.2.1.3) refers to be catalyzed and goes back from oligosaccharide and the non-of polysaccharide molecule The enzyme of originality end release D-Glucose.Glucoamylase is considered as generally also a type of amylase, referred to as starch-Portugal Glycosidase.

As used herein, term " amylase " (EC3.2.1.1) refer to by with inscribe or circumscribed model of action hydrolyzing alpha- 1,4 and/or α-1,6 glucoside bonds carry out the starch lyases of degradable starch and related compound.Amylase includes α-amylase (EC3.2.1.1)；Beta amylase (3.2.1.2), starch-amylase (EC3.2.1.3), alpha-Glucosidase (EC3.2.1.20), Amylopectase (EC3.2.1.41) and isoamylase (EC3.2.1.68).In some embodiments, amylase is alphalise starch Enzyme.

As used herein, term " pectase " refers to be catalyzed the unit that hydrolyzed pectin becomes less, such as oligosaccharide or monomer sugar Enzyme.In some embodiments, enzymatic mixture includes any pectase, such as inscribe-polygalacturonase, pectin methyl Esterase, inscribe-Galactanase, pectin acetyl base esterase, inscribe-pectin lyase, transelminase, α rhamnosidase, Circumscribed-galacturonic acid enzyme, circumscribed-galacturonan lyase, rhamnosan galacturonic acid (rhamnogalacturonan) hydrolytic enzyme, rhamnosan polygalacturonate lyase, rhamnosan galacturonic acid acetyl group Esterase, rhamnosan galacturonic acid galacturonic hydrolase and/or wood galacturonic acid enzyme (xylogalacturonase).

As used herein, term " inscribe-polygalacturonase " (EC3.2.1.15) refer to be catalyzed pectic acid and other 1,4-α-D-galactoside aldehydic acid key (1,4-α-D-in galacturonic acid polysaccharide (galacturonan) Galactosiduronic linkage) the enzyme of random hydrolysis.This enzyme is also referred to as " polygalacturonase pectin Depolymerase ", " pectase (pectinase) ", " inscribe polygalacturonase ", " pectase (pectolase) ", " pectin water Solve enzyme ", " polygalacturonase ", " poly-α-1,4-galacturonic acid glycosides endohydrolase ", " inscribe galacturonic acid Enzyme ", " inscribe-D-galacturonic acid enzyme " or " poly-(1,4-α-D-galacturonic acid glycosides) endohydrolase ".

As used herein, term " pectin methylesterase " (EC3.1.1.11) refers to be catalyzed the enzyme of following reaction: pectin+n H₂O=n methanol+pectic acid.This enzyme can also be referred to as " pectinesterase ", " PE (pectin Demethoxylase) ", " PE ", " pectin methylesterase ", " pectase ", " pectinesterase " or " pectinesterase hydrolytic enzyme (pectin pectylhydrolase) " (pectinoesterase).

As used herein, term " inscribe-Galactanase " (EC3.2.1.89) refers to be catalyzed in arabinogalactan The enzyme of endo hydrolysis (endohydrolysis) of 1,4-β-D-galactose glycosidic bond.This enzyme can also be referred to as " Arabic gala Polysaccharide inscribe-1,4-beta galactosidase ", " inscribe-1,4-paragalactan enzyme ", " Galactanase ", " arabinogalactan Carbohydrase " or " arabinogalactan 4-β-D-galactan hydrolytic enzyme ".

As used herein, term " pectin acetyl base esterase " refers to the acetyl group being catalyzed at the hydroxyl of pectin GaIUA residue The enzyme of deacetylation.

As used herein, term " inscribe-pectin lyase " (EC4.2.2.10) refers to be catalyzed (1 → 4)-α-D-galactose Aldehydic acid polysaccharide methyl ester elimination cutting with produce its non reducing end have 4-deoxidation-6-O-methyl-α-D-gala- The enzyme of the oligosaccharide of 4-olefine aldehydr acid glycosyl (4-deoxy-6-O-methyl-α-D-galact-4-enuronosyl) group.This enzyme is also It is properly termed as " pectin lyase ", " Pectin trans-elimination enzyme (pectin trans-eliminase) ", " inscribe-pectin lyase Enzyme ", " poly-methyl galacturonate trans elimination enzyme ", " pectin methyl trans elimination enzyme ", " pectin lyase (pectolyase) ", " PL ", " PNL ", " PMGL " or " (1 → 4)-6-O-methyl-α-D-galacturonic acid polysaccharide lyase ".

As used herein, term " transelminase " (EC4.2.2.2) refers to be catalyzed (1 → 4)-α-D-galacturonic acid The elimination cutting of polysaccharide has 4-deoxidation-α-D-gala-4-olefine aldehydr acid glycosyl (4-deoxy-to produce at non reducing end α-D-galact-4-enuronosyl) enzyme of oligosaccharide of group.This enzyme can also be referred to as " polygalacturonic acid trans elimination enzyme (polygalacturonic transeliminase) ", " pectic acid trans elimination enzyme ", " galacturonan lyase ", " inscribe pectin methyl trans elimination enzyme ", " pectic acid trans elimination enzyme ", " inscribe galacturonic acid trans elimination enzyme ", " pectin Acid cleavage enzyme ", " pectin lyase ", " α-1,4-D-inscribe galacturonan lyase ", " PGA lyases ", " PPase- N ", " inscribe-α-1,4-galacturonan lyase ", " galacturonan lyase ", " Pectin trans elimination enzyme ", " poly- Galacturonic acid trans elimination enzyme " or " (1 → 4)-α-D-galacturonic acid polysaccharide lyase ".

As used herein, term " α-rhamnosidase " (EC3.2.1.40) refers to be catalyzed alpha-L-rhamnoside or optional The enzyme of the hydrolysis of the end irreducibility α-L-rhamnose residue in rhamnosan galacturonic acid.This enzyme can also be referred to as " alpha-L-Rhamnosidase T ", " alpha-L-Rhamnosidase N " or " alpha-L-rhamnoside rhamnose hydrolytic enzyme ".

As used herein, term " circumscribed-galacturonic acid enzyme " (EC3.2.1.82) refers to hydrolyze from non reducing end Pectic acid, the enzyme of release digalactosyl aldehydic acid (digalacturonate).This enzyme can also be referred to as " circumscribed-poly-α-galactose Aldehyde neuraminidase ", " circumscribed poly-tilactase " or " circumscribed polygalacturonase ".

As used herein, term " circumscribed-galacturonic acid polysaccharide Isosorbide-5-Nitrae-α galacturonic acid neuraminidase " (EC3.2.1.67) is The enzyme of the finger following type reaction of catalysis: (1,4-α-D-galacturonic acid neuraminidase) n+H2O=(1,4-α-D-galacturonic acid glycosides) n- I+D-galacturonic acid.This enzyme can also be referred to as " poly-[1-> 4) α-D-galacturonic acid glycosides] galacturonic hydrolase ", " outer Cut polygalacturonase ", " poly-(galacturonic acid) hydrolytic enzyme ", " circumscribed-D-galacturonic acid enzyme ", " circumscribed-D-galactose Aldehydic acid enzyme ", " circumscribed poly-D-galacturonic acid enzyme " or " poly-(1,4-α-D-galacturonic acid glycosides) galacturonic hydrolase ".

As used herein, term " exopolygalacturonate lyase " (EC4.2.2.9) refer to be catalyzed 4-(4-deoxidation- α-D-gala-4-olefine aldehydr acid glycosyl)-D-galacturonic acid is from the elimination of the reducing end under neutral of pectic acid (i.e. the pectin of deesterify) Property cutting enzyme.This enzyme is also referred to as " pectic acid disaccharide lyases ", " the circumscribed lyases of pectic acid ", " circumscribed pectic acid is anti- Formula eliminate enzyme ", " circumscribed transelminase ", " circumscribed polygalacturonic acid-trans-elimination enzyme ", " PATE ", " circumscribed- PATE ", " circumscribed-PGL " or " (1 → 4)-α-D-galacturonic acid polysaccharide reducing end under neutral disaccharide lyases ".

As used herein, term " rhamnosan galacturonic acid enzyme " refer to by disaccharide [(1,2-α-L-rhamnose acyl- (1,4)-α-galactosyl aldehydic acid] in the strict rhamnosan galacturonic acid structure alternately that forms with internal-cutting way hydrolysis half The enzyme of the key between lactose base aldehydic acid and rhamnopyranosyl.

As used herein, term " rhamnosan polygalacturonate lyase " refers to borrow in rhamnosan galacturonic acid Help β-elimination with the enzyme of internal-cutting way cutting α-L-Rhap-(1 → 4)-α-D-GalpA key.

As used herein, term " rhamnosan galacturonic acid acetyl esterase " refers to be catalyzed rhamnosan galacturonic acid The enzyme of the alternately deacetylation of main chain of rhamnose and galacturonic acid residues in acid.

As used herein, term " rhamnosan galacturonic acid galacturonic hydrolase " refer to beyond butt formula hydrolysis The enzyme of galacturonic acid from the non reducing end of strict rhamnosan galacturonic acid structure alternately.This enzyme also may be used To be referred to as " wood galacturonic acid polysaccharide (xylogalacturonan) hydrolytic enzyme ".

As used herein, term " inscribe-arabanase " (EC3.2.1.99) refers to be catalyzed 1,5-arabinan In the enzyme of endo hydrolysis of 1,5-α-arabinofuranosidase glycosidic bond.This enzyme can also be referred to as " inscribe-Arab's enzyme ", " I Primary polysaccharide inscribe 1,5-α-L-arabinose glycosides enzyme ", " inscribe-1,5-α-L-arabanase (endo-1,5-α-L- Arabinanase) ", " inscribe-α-1,5-L-arabanase (endo-α-1,5-L-arabinase) ", " inscribe-I Primary dextranase " or " 1,5-α-L-arabinan 1,5-α-L-arabinose hydrolytic enzyme ".

As used herein, " protease " includes the enzyme (peptidase) of hydrolysising peptide key and range of hydrolysed peptides and other parts such as sugar Between the enzyme (glycopeptidase) of key.Many protease are characterized under EC3.4 and are suitable to use in the present invention.Some are specific The protease of type includes but not limited to, cysteine proteinase includes pepsin, papain, and serine protease Including Chymotrypsin, carboxypeptidase, and metal endopeptidase.

As used herein, " lipase " includes hydrolyzing lipid, fatty acid and acyl glyceride, including phosphoglyceride, fat egg In vain, the enzyme of DG etc..In plant, lipid is used as limiting moisture loss and the structural constituent of pathogenic infection.This A little lipids include the wax from fatty acid derived and cutin and suberin.

As used herein, term " separation " and " purification " be used to refer to by from its natural association at least one other Molecule (such as, the nucleic acid of separation, polypeptide [including but not limited to enzyme], etc.) that component is taken out or other components.Therefore, these arts Language refers to the material taken out from its primal environment (such as, natural surroundings, if it is to naturally occur).Expect this term Any suitable method including at least one component for removing the natural association of this molecule.In some embodiments, this A little terms also include the cell separated from other cells and/or nutrient media components.Expect that any suitable separation method can be used In the present invention.In some embodiments, when material is with ratio present in naturally occurring organism or wild-type organisms When higher or lower concentration is present in particular composition, or its with by naturally occurring organism or wild-type biology body surface During the component combination that there is usually no after reaching, this material is expressed as " purification ".Such as, natural present in the animal of survival The polynucleotide or the polypeptide that exist are unsegregated, but are separated in the some or all of coexisting materials from natural system Same polynucleotide or polypeptide be separate.In some embodiments, these polypeptide are parts for carrier, and/or These polynucleotide or polypeptide are parts for compositions, and are still considered as separation, because described carrier or compositions are not It it is a part for its natural surroundings.In some embodiments, nucleic acid or protein are expressed as purification, such as, if its If producing substantially one band in running gel or trace.

When being used for representing DNA sequence, term " separation " refer to by from its natural genetic environment take out and the most not Containing other unrelated or undesired coded sequences, and it therefore it is the form being suitable for use in the protein production systems of genetic modification DNA sequence.These molecules separated by the molecule separated from its natural surroundings and include cDNA and genomic clone.Though Other genes that so DNA molecular of the separation of the present invention generally associates without it, but can include that naturally occurring 5' and 3' is non- Translated region (such as, promoter and terminator).The qualification of association region will be apparent from (ginseng for those of ordinary skill in the art See such as, Dynan and Tijan, Nature316:774-78 [1985]).Selectively, term " DNA sequence of separation " is claimed For " DNA sequence of clone ".

When being used for representing protein, term " separation " refers to the egg found in the condition in addition to its natural surroundings White matter.In some embodiments, the protein of separation is substantially free of other protein, particularly other homologous proteins.As Being determined by SDS-PAGE, the protein of separation is greater than about 10% pure, and preferably more than about 20% is pure and the biggest Pure in about 30%.As determined by SDS-PAGE, the other aspect of the present invention includes the purest form (i.e., greater than about 40% Pure, greater than about 60% pure, greater than about 70% pure, greater than about 80% pure, greater than about 90% pure, greater than about 95% pure, greater than about 97% pure, big Pure or even greater than about 99% pure in about 98%) protein.

When being used for representing cellobiose dehydrogenase, " purification " or " separation " refer to by means of some originally associated from it or The composition that all natural exists separates cellobiose dehydrogenase and makes cellobiose dehydrogenase change from its native state.This can By any suitable method, including art-recognized isolation technics, include but not limited to ion-exchange chromatography, affine layer Analysis, hydrophobic separation, dialysis, Protease Treatment, ammonium sulfate precipitation or other albumen salt precipitations, centrifugal, size exclusion chromatography, mistake Filter, microfiltration, gel electrophoresis, gradient separations or any other suitable method complete, less desirable to remove in final composition Intact cell, cell debris, impurity, irrelevant protein or enzyme.Then by the composition of benefit other for offer, such as activator, Counter inhibitor, desired ion, control the compound of pH, other enzymes etc. and add in the compositions comprising cellobiose dehydrogenase also It is possible.

As used herein, phrase " the purest polypeptide " refer to wherein this peptide material be existing main matter (i.e., By mole or weight in terms of, it is more more rich than any other single macromolecular substances in compositions) compositions, and work as purpose Material by mole or % weight in terms of macromolecular substances existing for composition at least about percent 50 time, it is typically the purest Compositions.Generally, the purest enzymatic compositions by mole or % weight in terms of by all macromolecular complex present in composition said composition About the 60% or more of matter, about 70% or more, about 80% or more, about 90% or more, about 95% or more or about 98% or more Many.Vehicle substance, little molecule (< 500 dalton) and the ionic species of element are not qualified as macromolecular substances.

As used herein, term is mentioned that " purifying process " that enzymatic mixture uses is contained and is physically removed enzymatic mixture not Expect any technique of component.Therefore, in some embodiments, purifying process provided herein includes from enzymatic mixture physics Ground removes cellobiose oxidase active or vice versa purification process as the same.Expect any suitable purification work known in the art Skill can be used for the present invention.It is true that do not expect that the present invention is limited to any specific purifying process.

As used herein, term " acellular enzymatic mixture " includes from any cell, including secreting the thin of this enzyme The enzyme that born of the same parents separate.Acellular enzymatic mixture can be prepared by any one of multiple method known in the art, such as filter or Centrifugal method.In some embodiments, it may be that such as, part is acellular, the most acellular or complete for enzymatic mixture The most acellular.

As used herein, " polynucleotide " refer to strand or the deoxyribonucleotide of double chain form or ribonucleotide Polymer and its complement (complement).

If polynucleotide are with its native state maybe when operating by method known to those skilled in the art, it can quilt Transcribing and/or translate to produce RNA, polypeptide or its fragment, the most described polynucleotide are expressed as " coding " RNA or many Peptide.The antisense strand of this nucleic acid is also expressed as encoding these sequences.As known in the art, the transcribed DNA of RNA polymerase is to produce Raw RNA, and the reversible RNA that transcribes of reverse transcriptase is to produce DNA.Therefore, on the contrary DNA molecular can effectively coding RNA molecule and As the same.

Term " albumen " and " polypeptide " are interchangeably used herein, refer to the polymer of amino acid residue.Additionally, art Both naturally occurring and synthetic aminoacid and amino acid analogue contained in language " aminoacid ", " polypeptide " and " peptide ".Naturally occur Aminoacid be those encoded by genetic code and those aminoacid the most adorned (such as, hydroxyproline, γ- Carboxyglutamic acid and O-phosphoserine).

As used herein, " destination protein " and " desired polypeptides " refers to protein/polypeptide that is desired and/or that be evaluated.? In some embodiments, destination protein is by intracellular expression, and in other embodiments, it is the polypeptide of secretion.Real at some Executing in scheme, " destination protein " or " desired polypeptides " includes the enzyme of the present invention.In some embodiments, destination protein is for be melted Close the polypeptide of the secretion of signal peptide (that is, the aminoterminal extension on albumen to be secreted).The albumen of nearly all secretion Matter uses and is being targeted to precursor protein and precursor protein through the amino-terminal protein matter extension played a crucial role in the transfer of film Point.This extension is removed by protease hydrolysis by signal peptidase during film shifts or after film transfer immediately.

As used herein, term " amino acid analogue " refers to have the substantially chemistry identical with naturally occurring aminoacid (that is, alpha-carbon atom is incorporated into hydrogen atom, carboxyl, amino and R base, includes but not limited to homoserine, the brightest for the compound of structure Propylhomoserin, methionine sulfoxide and methionine methyl sulfonium).In some embodiments, these analog have the R base of modification (such as, nor-leucine) and/or the peptide backbone modified, but retain the basic chemical structure identical with naturally occurring aminoacid.

Aminoacid is at three letter symbols or the list of IUPAC-IUB biochemical nomenclature commission suggestion known to it herein Alphabetic character is mentioned.Similarly, nucleotide can be mentioned by its generally accepted single letter code.

" position " of aminoacid or nucleotide base by according to it relative to N-terminal (or 5 ' ends)

Sequence of positions distinguish that the numbering of each aminoacid (or nucleotide base) represents.Owing to determining optimal comparison Disappearance that Shi Bixu is considered, insertion, truncate, fusion etc., the cycle tests determined by counting from N-terminal simply Numbering amino acid residues is not necessarily identical with the numbering of its correspondence position in reference sequences.Such as, have at cycle tests In the case of the disappearance of the reference sequences of comparison, variant there will be no corresponding in reference sequences at deletion segment The aminoacid of position.Existing in the case of inserting in the cycle tests of comparison, this insertion will not be corresponding in reference sequences The amino acid position of numbering.In the case of truncate or merging, can exist in reference sequences or aligned sequences and not correspond to Any amino acid whose aminoacid string (stretches of amino acids) in corresponding sequence.

It is used interchangeably term " wild-type sequence " herein and " naturally occurring sequence " represents intrinsic in host cell Or naturally occurring polypeptide or polynucleotide sequence.In some embodiments, wild-type sequence is expressed as protein engineering item The aim sequence of purpose starting point.Wild-type sequence codified homology or heterologous protein.Host cell when homologous protein is without intervening The protein produced.Heterologous protein is unless intervened the protein that otherwise host cell will not produce.

As used herein, " naturally occurring enzyme " represents aminoacid sequence (that is, " open country having with discovery in nature Raw type ") enzyme of identical the most adorned aminoacid sequence.Naturally occurring enzyme includes constitutive enzyme (that is, sky in specified microorganisms Those enzymes so expressed or find).

As used herein, term " reference enzyme " refer to the another kind of enzyme (such as, " testing " enzyme) of the present invention compare with it with Determine the enzyme of the existence of the feature of improvement in other enzymes being evaluated.In some embodiments, reference enzyme is wild-type enzyme. In some embodiments, reference enzyme refers to that the test enzyme of the present invention compares with it to determine the improvement in the test enzyme being evaluated The enzyme of existence of feature, thermal activities that the character of described improvement includes but not limited to improve, the heat stability of improvement and/or The stability improved.In some embodiments, reference enzyme is wild-type enzyme.

As used herein, term " bioactive fragment " refers to have amino terminal and/or carboxyl-terminal deletion and/or interior The polypeptide of the disappearance in portion, but in the sequence that wherein remaining aminoacid sequence compares with it, relevant position is identical, and this polypeptide Retain the essentially all activity of full-length polypeptide.

As used herein, term " is recombinated " and is referred to polynucleotide or the polypeptide of non-natural existence in host cell.At some In embodiment, recombinant molecule comprises the sequence of the two or more naturally occurrings linked together in the way of non-natural existence Row.In some embodiments, due to deliberate manual intervention, " reconstitution cell " expresses natural (that is, non-recombinant) at cell Form does not finds the gene of same form and/or expresses original process LAN, low expression and/or the sky the most do not expressed singularly So gene.Reconstitution cell comprises polynucleotide or the polypeptide of at least one restructuring.Nucleic acid construct, nucleic acid (the most nucleoside Acid), polypeptide or host cell when be non-naturally occurring, artificial or engineered time, be referred to herein as " restructuring ". " restructuring (Recombination) ", " restructuring (recombining) " and generation " restructuring " nucleic acid generally contain at least two core The assembling of acid fragment.The present invention also provides for the nucleic acid construct of restructuring, is included under stringent hybridization condition and has SEQ with coding At least one CDH polynucleotide sequence of the complement hybridization of the polynucleotide of the polypeptide of the aminoacid sequence of ID NO:2.

When nucleic acid associates, and when associating the most in the solution, they " hybridize ".Nucleic acid hybridization is to be characterized very well due to various Physical-chemical power, such as hydrogen bonding, Solvent Repellency (solvent exclusion), base stacking etc..It is as used herein, Term " strict hybridization washes " under the background of nucleic acid hybridization test such as Southern hybridization or Northern hybridization It is sequence dependent, and is different under varying environment parameter.Extensively instructing of nucleic acid hybridization is seen Tijssen, 1993,"Laboratory Techniques in Biochemistry and Molecular Biology- Hybridization with Nucleic Acid Probes, " it leads to for part i, the 2nd chapter (Elsevier, New York) Cross and be incorporated herein by reference.For the polynucleotide of at least 100 length of nucleotides, to low stringency condition to high strict bar Part is defined below: after the southern blotting technique program of standard, shears and degeneration at 5 × SSPE, 0.3%SDS, 200 μ g/ml at 42 DEG C Salmon sperm dna, and 25% Methanamide for low stringency, for medium and in-35% Methanamide of high stringency, or for Prehybridization and hybridization in 50% Methanamide of high stringency and high stringency.Many nucleoside at least 100 length of nucleotides Acid, uses 2 × SSC, 0.2%SDS50 DEG C (low stringency), 55 DEG C (medium stringency), 60 DEG C (in-high stringency), 65 DEG C (high stringency) or 70 DEG C (high stringency), carrier material is finally washed three times, each 15 minutes.

Medium stringency condition contains known in the art and those described in the multiple standards textbook, and includes washing molten Liquid and the use of hybridization conditions (such as, temperature, ionic strength and %SDS).One example of medium stringency condition includes: 37 Overnight incubation DEG C in comprising following solution: 20% Methanamide, 5x SSC (150mM NaCl, 15mM trisodium citrate), 50mM The salmon sperm DNA that sodium phosphate (pH7.6), 5x Denhardt solution, 10% dextran sulfate and 20mg/mL degeneration are sheared, exists subsequently About 37-50 DEG C washing nozzle in 1x SSC.Those skilled in the art know how to adjust as required temperature, ionic strength etc. To adapt to the factor of such as probe length and similar factor.

Such as use in some embodiments herein, stringent condition or high stringent conditions use: (1) low ion is strong Degree and high temperature are used for washing, such as at 50 DEG C of 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulphate； (2) during hybridization, denaturant such as Methanamide, such as, 50% (v/v) Methanamide and 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer, pH6.5,750mM sodium chloride, 75mM sodium citrate, At 42 DEG C；Or (3) 50% Methanamides, 5x SSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5x Denhardt solution, salmon sperm DNA (50 μ g/mL), 0.1%SDS and the 10% sulphuric acid Portugal of supersound process gather Sugar, at 42 DEG C, 42 DEG C in 0.2x SSC (sodium chloride/sodium citrate) and 55 DEG C in 50% Methanamide wash, exist subsequently 55 DEG C of high stringency washings formed with the 0.1x SSC containing EDTA.

As used herein, the term " mutant library " and " Mutant libraries " for representing cell refers to their genome But major part is identical includes the cell colony of the different congener of one or more gene.These libraries can be used to, Such as identify gene or the operon of the character with improvement.When being used for representing polypeptide or nucleic acid, " library " refers to one group (i.e., Multiple) heterologous polypeptide or nucleic acid.Library is made up of " member ".Sequence difference between the member in library causes in library and exists Multiformity.The form of the simple mixtures of polypeptide or nucleic acid can be taked in library, can be maybe the biology converted by nucleic acid library Body or cell, such as antibacterial, virus, the form of animal or plant cell and similar type.

As used herein, term " initial gene " refers to that the present invention to be used encoding destination protein improves, lacks, suddenlys change And/or the genes of interest otherwise changed.

Term " character " as used by herein under nucleic acid background and grammatical equivalents thereof thereof refer to the core that can be chosen or detect Any feature of acid or attribute.These character include but not limited to affect the character of Binding peptide, imparting comprises specific nucleic acid The character of cell, affect character (such as, promoter intensity, Promoter Recognition, promoter regulation and/or the enhancing of genetic transcription Subfunction), affect RNA processing character (such as, RNA montage, rna stability, RNA conformation and/or posttranscriptional modification), impact The character (such as, level, regulation and control, the combination of mRNA and ribosomal protein and/or post translational modification) of translation.Such as, can change Become the nucleic acid binding site to transcription factor, polymerase, regulatory factor etc. to produce desired feature or to identify less desirable Feature.

Term " character " as used by herein under polypeptide (including protein) background and grammatical equivalents thereof thereof refer to can be selected Any feature of the polypeptide selected or detect or attribute.These character include but not limited to oxidation stability, substrate specificity, catalysis Activity, heat stability, alkaline stability, pH activity curve, the resistance to proteasome degradation, k_m、k_cat、k_cat/k_mRatio, protein folding Fold, induce immunoreation, do not induce immunoreation, the ability of binding partner, the ability of bind receptor, secreted ability, quilt The ability that is illustrated on cell surface, the ability of oligomerization, the ability of signal transduction, the ability stimulated cellular proliferation, suppression are thin Born of the same parents propagation ability, apoptosis-induced ability, by by phosphorylation or glycosylation modified ability and/or treatment disease ability Deng.It practice, expection the invention is not restricted to any specific character.

As used herein, " similarity " refers to that identical or its conserved amino acid replaces, as defined below.Therefore, in order to The purpose of similarity, changes into identical or conservative and replaces and be not intended as including changing.Amino acid whose disappearance or nonconserved amino acid It is substituted in and is considered as herein including changing.The calculating of Similarity Percent is carried out with the same way carrying out homogeneity percentage ratio.

As used herein, about representing that " conservative replacement " used by aminoacid refers to the aminoacid replacement being chemically similar A kind of aminoacid.The general aminoacid replacement not changing specific activity is well known in the art and is described in many textbooks. The exchange most commonly occurred be Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/ Gly and the reverse exchange of these exchanges.As used herein, another during the conservative substitutions for a kind of residue is same group A kind of residue.

In some embodiments, conserved amino acid replaces can be the such as substituted replacement of conservative shown in Table A. The replacement of display is based on amino acid whose physicochemical properties, so, is independently of organism.In some embodiments, It is replacement listed under exemplary replacement title that conserved amino acid replaces.

As used herein, when use in the context that given aminoacid or peptide sequence are numbered term " reference ... Numbering " or refer to when this given aminoacid or peptide sequence are compared with reference sequences with specific reference to sequence time " corresponding to ... " The numbering of residue.

Hereinafter name can be used to the replacement describing in reference sequences relative to reference sequences or variant polypeptide or nucleotide sequence: " R-#-V ", wherein # refers to the position in reference sequences, and R refers to the aminoacid (or base) of this position in reference sequences, and V Refer to the aminoacid (or base) of this position in variant sequence thereof.

Term " aminoacid replacement set " and " replacing set ", refer to one group of aminoacid replacement.One replace set can comprise 1, 2,3,4,5,6,7,8,9,10,11,12,13,14,15 or more kinds of aminoacid replacement.

As used herein, in two or more peptide sequence contexts, " homogeneity " or " homogeneity percentage ratio " refers to as utilized Sequence alignment algorithms or by manpower comparing to and macroscopic examination measured by, when across comparison window or across specify region carry out maximum When consistent contrast and comparison, two or more sequences or subsequence are being identical with reference sequences across appointment region or are having The same amino acid residue percentage ratio specified (such as, total at least about 70%, at least about 75%, at least about 80%, at least about 85%, At least about 88% homogeneity, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, At least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homogeneity).In some embodiments, art Language " homogeneity percentage ratio (percent identity) ", " % homogeneity ", " same percentage ratio (percent identical) " " % identical (%identical) ", used interchangeably herein, referring to (can be from European by ClustalW analysis Bioinformatics Institute, the W1.8 version that Cambridge, UK obtain) aminoacid that obtains or polynucleotide sequence Homogeneity percentage ratio: calculate the number of identical match in comparison and with the number of this identical match divided by the length of reference sequences, And use following ClustalW parameter to reach slowly/to penalize by most preferably open than p-DNA/ albumen room more accurately Divide (Gap Open Penalty): 15/10；DNA/ albumen gap extension penalties (Gap Extension Penalty): 6.66/ 0.1；Albumen weight matrix (Protein weight matrix): Gonnet series；DNA weight matrix: Identity.

Two sequences is " comparison " when following time: use the amino acid substitution matrix (example limited for similarity score Such as, BLOSUM62), room exist point penalty and gap extension penalties this two sequences is compared reaches this sequence pair may Highest score.Application in amino acid substitution matrix and the similarity between quantitative two sequences thereof is well known in the art (see for example, Dayhoff et al., in Dayhoff [writing],Atlas of Protein Sequence and Structure, " volume 5, supplementary issue 3, Natl.Biomed.Res.Round., Washington D.C. [1978];pp.345- In 352；With Henikoff et al., Proc.Natl.Acad.Sci.USA, 89:10915-10919 [1992], both is by drawing With being expressly incorporated herein).BLOSUM62 matrix is typically used as default scoring in alignment scheme such as Gapped BLAST2.0 and takes For matrix (default scoring substitution matrix).There is point penalty for drawing in one of aligned sequences in room Enter single amino acids room, and gap extension penalties is empty for each other aminoacid being inserted in open room Position.Started and terminate the amino acid position of every sequence at place by comparison and optionally by one or two sequences In row, one room of insertion or multiple room are to reach highest possible scoring, limit comparison.Although optimal comparison and scoring can Manually complete, but promote this process (such as, gapped BLAST2.0 by the alignment algorithm using computer to perform；See, Altschul et al., Nucleic Acids Res., 25:3389-3402 [1997], be incorporated herein by), and this calculation Method in US National biotechnology center (National Center for Biotechnology Information) website is Publicly available.The program being readily available such as PSI-BLAST can be used (to see for example, Altschul et al., above), prepare Good comparison, including multiple ratio pair.

The present invention also provides for the nucleic acid construct of restructuring, and this construct is included under stringent hybridization condition to be had with coding The CDH polynucleotide sequence of the complement hybridization of the polynucleotide of the polypeptide of the aminoacid sequence of SEQ ID NO:2.Have 100% Two nucleic acid of sequence iden or peptide sequence are referred to as " identical ".When such as using methods described herein, such as use standard The BLAST of parameter determines, nucleic acid or peptide sequence and reference sequences have at least about 70%, at least about 75%, at least about 80%, At least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% or during bigger sequence iden, nucleic acid or peptide sequence are referred to as and ginseng Examine sequence and there is " substantially sequence iden ".

As used herein, " secreting signal peptide " can be former peptide (propeptide), propetide (prepeptide) or the two. Such as, term " former peptide " refers to the amyloid protein precursor of cleaved generation " maturation protein ".Before secretion, signal peptide is by signal peptidase In the past protein cleavage, produces " ripe " or " secretion " albumen.Term " propetide " and " front albumen " refer to be synthesized into target Polypeptide to its N-terminal signal peptide for secreting.Therefore, " front-former-peptide " is to comprise target polypeptide for secreting and cleaved Under to produce the polypeptide of signal peptide of mature polypeptide.Signal peptide sees the N-terminal of albumen, generally by 6 to 136 alkalescence and hydrophobic Amino acid profile.

As used herein, " transcribing " and similar terms refer to that encoding information in transformed gene is rna transcription.Correspondingly, The transcriptional level of minimizing cellobiose oxidisability enzyme is subtracting of the amount of rna transcription of the RNA of encoding fiber disaccharide oxidisability enzyme Few.

As used herein, term " DNA construct " and " convertibility DNA " are employed interchangeably to represent for by sequence It is incorporated into the DNA in host cell or organism.DNA can pass through PCR or well known by persons skilled in the art, and any other is suitable The external preparation of technology.In some embodiments, DNA construct comprises aim sequence (such as, as " sequence of introducing "). In some embodiments, described sequence is operably coupled to other element and such as controls element (such as, promoter etc.). In some embodiments, DNA construct also includes at least one optional mark.In some other embodiments, DNA Construct comprises the calling sequence that flank is homology frame.In some other embodiments, convertibility DNA comprises and is added to two Other nonhomologous sequence (such as, padding sequence or flank) of individual end.In some embodiments, the end of calling sequence is Guan Bi so that convertibility DNA forms the ring of Guan Bi.Convertibility sequence can be wild type, sudden change or modify.At some In embodiment, DNA construct comprises the sequence of the homology of chromosome with host cell.In some other embodiments, DNA Construct comprises nonhomologous sequence.Once DNA construct is by assembled in vitro, then it can be used to: 1) heterologous sequence is inserted into place In the desired target sequence of chief cell；2) region mutagenesis of host cell chromosome (that is, is replaced endogenous sequence with heterologous sequence Row)；3) target gene is removed；And/or 4) replicability plasmid is incorporated in host.In some embodiments, calling sequence bag Containing at least one optional mark.These one or more destination proteins of sequence codified.It can have other biological function.Permitted In the case of Duo, calling sequence comprises at least one optional mark, such as, provide the gene of antimicrobial resistance.

As used herein, " carrier " is the polynucleotide constructs for introducing polynucleotide sequence in cell.One In a little embodiments, carrier comprises and is operably connected to polynucleotide sequence being capable of in suitable host and expresses many The suitable control sequence of nucleotide sequence coded polypeptide." expression vector " has and is operably connected to polynucleotide sequence The promoter sequence that (such as, transgenic) expresses in host cell with driving, and in some embodiments, transcription terminator Sequence.In some embodiments, carrier is missing from carrier.In some embodiments, carrier comprises generation interference target multinuclear The siRNA of the translation of nucleotide sequence or the polynucleotide sequence of antisense RNA transcripton.

As used herein, " deleted carrier " comprises and the polynucleotide of the target sequence 5 ' and 3 ' treated from host genome disappearance The polynucleotide sequence of sequence homology, thus instruct with the polynucleotide restructuring between 5 ' and 3 ' targeting sequences and replace target sequence Row.

As used herein, any step involved by the generation including polypeptide " expressed " in term, includes but not limited to: turns Record, posttranscriptional modification, translation and post translational modification.In some embodiments, this term is also contemplated by from cell secretory polypeptide. Generally, this term " is expressed " and is referred to that the information that transformed gene encodes is by the albumen of this gene code.Therefore, " the fiber of expression The minimizing of the amount of disaccharide oxidisability enzyme " it is the minimizing of the amount of the cellobiose oxidisability enzyme finally translated by this cell.

As used herein, term " process LAN " is intended to include the expression of albumen is increased to greater than the water that cell normally produces Flat.It is intended to the process LAN making this term include intrinsic protein and heterologous protein.In some embodiments, process LAN include with The endogenous transcription rate (transcription rate) of gene and/or level are compared, the transcription rate of the rising of this gene and/or water Flat.Such as, in some embodiments, heterologous gene is introduced into fungal cell to express encoding heterologous enzyme such as from another kind The gene of the β-glucosyl enzym of organism.In some other embodiments, heterologous gene is introduced into fungal cell with process LAN The gene of encoding homologous enzyme such as β-glucosyl enzym.

In some embodiments, heterologous gene is to be modified with the gene of process LAN gene outcome.Implement at some In scheme, " process LAN " refers to wherein compared with the endogenous expression rate of gene (expression rate) and/or level, this base Because being caused the rate to raise and/or any state of horizontal expression.In some embodiments, process LAN includes and gene Endogenous translation rate (translation rate) and/or level are compared, the translation rate of the rising of this gene and/or level.

As used herein, term " produces " and refers to be produced albumen and/or other compounds by cell.Expect that this term is contained Any step involved in the generation of polypeptide, includes but not limited to, transcribe, posttranscriptional modification, translation and post translational modification.One In a little embodiments, this term is also contemplated by from cell secretory polypeptide.

As used herein, it is by described cell " by the cellobiose dehydrogenase of emiocytosis " so that described fiber two Glucocorticoid dehydrogenase exports through cell membrane and is then followed by being discharged into extracellular environment, and the mode being such as discharged in culture medium produces Cellobiose dehydrogenase.

As used herein, " being already adapted to the polynucleotide sequence expressed " is to have been inserted in expression vector or with other Mode is modified to be included in host cell the polynucleotide sequence expressing the required regulating element of polynucleotide, described regulation The mode that element is placed allows to express polynucleotide in host cell.Regulating element needed for this type of is expressed includes opening Promoter sequences, transcriptional initiation sequence and optionally enhancer sequence.Such as, in some embodiments, polynucleotide sequence quilt Insert in the plasmid being suitable to express in fungal host cells.

As used herein, term " plasmid " refers to annular double-strand (ds) DNA construct as cloning vehicle.Real at some Executing in scheme, plasmid forms the genetic elements of extrachromosomal self-replication in some eukaryotic cells and/or prokaryotic cell, and In some other embodiments, in plasmid integration to the chromosome of host cell.

As used herein, " control sequence " include for express present disclosure polynucleotide institute must or favourable All parts.Each control sequence can be intrinsic or external for polynucleotide of interest.These control sequences include but It is not limited to lead, polyadenylic acid tracts, propeptide sequence, promoter, signal peptide sequence and transcription terminator.

As used herein, " it is operatively connected " and refers to configure as follows: wherein control sequence and arranged suitably and (that is, be in In functional cohesion) in a position relative to polynucleotide of interest so that described control is sequence-directed or regulation and control purpose is many Nucleotide and/or the expression of polypeptide.

As used herein, when nucleic acid is arranged to the most relevant to another kind of nucleotide sequence, it is " operable company Connect ".Such as, if before the DNA of Encoding Secreted lead (that is, signal peptide) is expressed as participating in the secretion of a peptide species Body protein, then this DNA is operably coupled to the DNA of described polypeptide；If promoter or enhancer affect coded sequence Transcribe, then it is operably coupled to this sequence；Or if ribosome binding site is placed as promoting translation, then it can be grasped It is connected to coded sequence.Generally, " being operatively connected " refers to that the DNA sequence being connected is continuous print, and, before secreted In the case of leading thing, it is continuous print and is in reading code phase (reading phase).But, enhancer needs not to be continuous print.Even Connected the connection at convenient restriction site to complete.If such site does not exists, then use according to conventional practice The oligonucleotide adapter of synthesis or joint.

As used herein, region before and after term " gene " refers to coded polypeptide and includes coding region and each The polynucleotide (such as, region of DNA section) of the intervening sequences (intron) between individual coding section (exon).

As used herein, " endogenous " or " homology " gene refers to the parent at host cell (such as, fungus or bacterial cell) The gene (including but not limited to wild type) found in this bacterial strain.As used herein, when comparing between nucleotide sequence, " homologous genes " (or " congener " gene) refer to from different but the most relevant species correspond to each other and mutually the same or Closely similar gene.This term include by species formed (that is, the development of new species) and separate gene (such as, directly to Homologous genes) and the gene (such as, paralogous gene) that separated by genetic copying.

As used herein, aminoacid or nucleotide sequence (such as, promoter sequence, signal peptide, terminator sequence etc.) when Being operably connected to when nature another sequence incoherent with it, this two sequences is " allos ".

As used herein, " isodynamic enzyme " refers to the enzyme encoded by " heterologous gene ".But, it is also contemplated that, heterologous gene encodes Endogenous or homology enzyme, as described herein.Generally, term " heterologous gene " refers to be not found in the parent bacterium of host fungal cell Gene presented in strain (including but not limited to wild type).Therefore, in some embodiments, heterologous gene is derivative The phorozoon or sexual generally acknowledged from the fungal cell of the species and this gene of expression being different from the fungal cell expressing this gene The gene of the species of type or taxonomy equivalent.In some embodiments, heterologous gene is the base that host fungal cell is endogenous The modified forms of cause, this endogenous gene is operated, is subsequently introduced into or converts entrance host cell.Such as, at some In embodiment, heterologous gene has interior source coding sequence, but has the modification to promoter sequence.Similarly, real at some Execute in scheme, the aminoacid sequence that heterologous gene is identical with endogenous gene, but have and codon is utilized or to non-coding The modification of region such as intron or a combination thereof.Such as, in some embodiments, heterologous gene comprises and repaiies coded sequence It is decorated with the non-wild type peptide of coding.In some other embodiments, heterologous gene has the promoter identical with parent strain Sequence, 5 ' and 3 ' untranslated region and coding region, but compared with the parent strain of host cell, it is positioned at another district of same chromosome In territory or on diverse chromosome.

As used herein, the term used in the background in insertion nucleotide sequence to cell " introduces " and refers to convert, turn Lead, engage, transfect and/or known in the art for inserting nucleotide sequence to any other the suitable method in host cell. The present invention is can be used for any suitable means in host cell for introducing nucleic acid.

As used herein, mention that the term that cell uses " converts " and " conversion " refers to have and be integrated into its genome In non-native nucleic acid sequence or there are the cells of the plasmid episomals kept through many generations.In some embodiments, term " turns Change " and " stably converting " refer to have non-natural (that is, the allos) polynucleotide sequence being integrated in its genome Or there is the cell being maintained the free plasmid passed at least twice.

As used herein, term " host cell " and " host strain " refer to comprise many nucleoside provided herein for expression The suitable host of the expression vector of acid sequence (such as, DNA).In some embodiments, host cell is to be converted Or transfect protokaryon or the eukaryotic cell of the carrier using recombinant technique known in the art to build.The host converted can replicate volume The code carrier of at least one target protein and/or express desired target protein.Additionally, mention that the cell of specific bacterial strain refers to bacterium The parental cell of strain and offspring and the derivant of genetic modification.The derivant of the genetic modification of parental cell includes comprising modification Genome or the progeny cell of plasmid episomal, the genome of described modification or plasmid episomal give such as, and antibiotic resists Property, the fermentation etc. of improvement.In some embodiments, host cell is steady to have improvement protein excretion, albumen by genetic modification Qualitative or the expression of albumen and/or secrete the feature of other performances desired.Such as, the acquisition that knocks out of Alp1 function lacks albumen The cell of enzyme.Knocking out of pyr5 function obtains the cell with pyrimidine deficiency phenotype.In some embodiments, host cell It is modified lack endogenous cellulase protein-encoding sequence or otherwise eliminate one or more endogenous cellulase Express.In some embodiments, expressing of one or more endogenous cellulase is suppressed to increase target fibers element enzyme Produce.Genetic modification can be by any suitable gene engineering and/or classical microbiological technique (such as, chemistry or UV Mutation and screening subsequently) realize.Use recombinant technique, the side that the yield of enzyme in organism or culture increases can be caused Formula introduces, lacks, suppresses or modified nucleic acid molecule.Such as, knocking out of Alp1 function obtains the cell lacking protease.Pyr5 merit Knocking out of energy obtains the cell with pyrimidine deficiency phenotype.In some genetic engineering methods, homologous recombination is used to pass through Internal selectively targeted gene causes target gene to modify the expression of the albumen to check coding.In a kind of alternative method, SiRNA, antisense and/or ribozyme technology can be used for inhibition of gene expression.

It is used interchangeably term " adorned sequence " herein and " adorned gene " includes naturally occurring to represent Nucleotide sequence disappearance, insert, replace or any other interrupt sequence.In some embodiments, adorned sequence Expression product be the protein (such as, if this modification is disappearance or the interruption of sequence) of truncate.Some embodiment party In case, the protein of truncate remains biological activity.In some alternate embodiment, the expression product of adorned sequence is The protein (such as, modification comprises the insertion to nucleotide sequence) increased.In some other embodiments, insert and cause cutting Short protein (such as, resulting in the termination codon period of the day from 11 p.m. to 1 a.m when insertion).Therefore, insertion may result in protein or the growth of truncate Protein as expression product.

As used herein, term " nucleotide sequence of sudden change ", " nucleotide sequence of sudden change " and " gene of sudden change " quilt It is used interchangeably to represent that there is the core that at least one codon occurred in the wild-type nucleotide sequences of host cell changes Nucleotide sequence.The expression product of the sequence of sudden change is the protein of the aminoacid sequence relative to wild type with change.One In a little embodiments, expression product has the Functional Capability (such as, the enzymatic activity of enhancing) of change.

As used herein, term " targeting randomization " refers to produce that wherein one or more positions have been randomized is multiple The process of sequence.In some embodiments, randomization is that (that is, all four nucleotide, A, T, G and C can occur completely Random site).In some alternate embodiment, the randomization of nucleotide is confined to the subset of four kinds of nucleotide.Targeting is random Change and can be applicable to encode one or more codons of the sequence of one or more destination proteins.As by randomized codon Randoming scheme determines, when expressing, the library of gained produces wherein one or more amino acid positions can comprise all 20 Plant amino acid whose mixture or the protein group of aminoacid subset.In some embodiments, targeting randomization the group produced The single member of body is different in terms of amino acid number due to the targeting of codon or radom insertion or disappearance.At some additionally Embodiment in, the aminoacid of synthesis is included in produced protein group.In some other embodiments, by Most of membership tables of the colony that targeting randomization produces reveal the big sequence homology with consensus sequence than initial gene.? In some embodiments, one or more destination proteins of this sequential coding.In some alternate embodiment, these protein has There is different biological functions.

As used herein, the one or more amino acid modified polypeptide referred to by removal reference polypeptide " is lacked ". Disappearance can include removing 1 or more aminoacid, 2 or more aminoacid, 3 or more aminoacid, 4 or more amino Acid, 5 or more aminoacid, 6 or more aminoacid, 7 or more aminoacid, 8 or more aminoacid, 9 or more Aminoacid, 10 or more aminoacid, 15 or more aminoacid or 20 or more aminoacid, at most constitute the ammonia of this polypeptide The 10% or most of base acid sum constitutes the 20% of the aminoacid sum of this polypeptide, keeps enzymatic activity simultaneously and/or keeps institute's engineering The character of the improvement of the cellobiose dehydrogenase changed.Disappearance may be present in interior section and/or the end section of polypeptide.At some In embodiment, disappearance includes continuous print fragment, and in other embodiments, it is discontinuous.

As used herein, " gene delection " or " deletion mutation " is at least some of sequence of the DNA wherein constituting gene The sudden change that row are lost.Therefore, " disappearance " of addressing nucleic acid is the something lost destroyed wholly or in part causing constituting the DNA sequence of gene Pass loss or the replacement of material.In some embodiments, it is contemplated to the disappearance completely or almost completely of gene order.So And, in order to reduce the activity of the endogenous cellobiose oxidisability enzyme secreted by fungal cell, deletion mutation need not be removed completely The complete genome sequence of cellobiose oxidisability enzyme.Such as, eliminate in the avtive spot of encoding fiber disaccharide oxidisability enzyme Aminoacid, encoding secretion signals or coding are at the active aspect of the endogenous cellobiose oxidisability enzyme secreted by fungal cell The excalation of one or more nucleotide of the another part of the cellobiose oxidisability enzyme worked.Any amount can be lacked Nucleotide, from a base to whole piece chromosome.Therefore, in some embodiments, term " lacks " and refers to remove coding Gene (such as, cdh1) required for specified protein.In this case, the bacterial strain with this disappearance can be described as " disappearance bacterium Strain ".

As used herein, " fragment " refers to have aminoterminal and/or c-terminus and/or internal disappearance compared with reference polypeptide Polypeptide, but the most remaining aminoacid sequence is identical with the correspondence position in reference sequences.Fragment could generally have total length The polypeptide of cellobiose dehydrogenase, about 80%, about 90%, about 95%, about 98% or about 99% of such as polypeptide SEQ ID NO:2.One In a little examples, the peptide sequence with wild type cellobiose dehydrogenase of non-naturally-occurring disclosed herein can include initial first sulfur Propylhomoserin (M) residue (that is, the M at position 1).But, it would be recognized by those skilled in the art that such as at host cell or external turn over Translating in system, this initial methionine residues can be removed in the biological processing of enzyme, lacks initial first sulfur ammonia to produce Acid residue, but remain the ripe enzyme of the character of this enzyme in other respects.Therefore, initial methionine is comprised for having The polypeptide of the cellobiose dehydrogenase each of disclosed herein of aminoacid sequence, present disclosure also includes having and has lacked The polypeptide of beginning methionine residues (that is, lacks the fragment of the cellobiose dehydrogenase polypeptide of methionine at position 1).

As used herein, " conditional mutation " be that there is under some environmental conditions wild type phenotype and at some other There is under part the sudden change of mutant phenotype.

As used herein, term " screens " and has its general sense in this area, usually multi-step process.First Step, it is provided that the nucleic acid of sudden change or the variant polypeptide from it.Second step, determines the nucleic acid of this sudden change or the character of variant polypeptide. 3rd step, by determined by character and the character of corresponding precursor nucleic acid, the character of corresponding naturally occurring polypeptide or initial material The Nature comparison of material (such as, initiation sequence) is to produce the nucleic acid of sudden change.For those skilled in the art it will be apparent that be used for Obtain and there is the nucleic acid of character of change or the screening sequence of protein depends on character and the expection mutant nucleic acid of parent material The modification that contributed to of generation.Therefore it will be understood by those skilled in the art that the invention is not restricted to treat screened any specific Character and hereafter description to character only list exemplary example.It is typically for screening the method for any special properties Described by this area.Such as, people can measure combination, optimum pH, specificity etc. before and after sudden change, wherein changes table Show change.In some embodiments, screening is carried out in high flux mode, including treating multiple samples of screening simultaneously, including but Do not limit and utilize chip, phage display, many substrates and/or the mensuration of indicator and/or known in the art any other is suitable Method.

As used in some embodiments, screening includes the selection step being enriched with purpose variant from variant group.This The example of a little embodiments include being chosen as host organisms give the variant of growth vigor and phage display or any its His methods of exhibiting, wherein can combine based on it or catalytic property capture variant from variant group.In some embodiments, variant Library is exposed to stressed condition (such as, being exposed to heat, protease or Denaturing).It follows that it is the completeest in evaluation and screening Whole variant or pass through selective enrichment.Expect that this term includes any suitable selection approach.It practice, the expection present invention does not limits In any specific screening technique.

As used herein, " genetic modification " and/or " cell of genetic modification " (such as, " fungus of genetic modification is thin Born of the same parents " and/or " fungal cell of genetic modification ") it is its hereditary material cell of having used that gene engineering changes.Heredity is repaiied The cell of decorations also refers to derivant or the offspring of the cell that its hereditary material used gene engineering to change.By genetic engineering skill The example of the genetic modification that art causes includes the modification to genomic DNA；The genetic modification caused by gene engineering another One example includes being incorporated into intracellular by stable heterologous nucleic acids.Such as, as provided herein, genetic modification provided herein Fungal cell is the fungal cell that its hereditary material has been changed, and it shifts gears is to reduce secreted cellobiose oxygen The activity of the oxydasis cellobiose secreted by the amount of the property changed enzymatic activity or minimizing.

In some embodiments, the DNA sequence of sudden change uses the site saturation mutagenesis of at least one codon to produce.? In some other embodiments, two or more codons are carried out site saturation mutagenesis.In some other embodiments In, the DNA sequence of sudden change and wild-type sequence have greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, be more than About 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95% or the homology of greater than about 98%.? In some alternate embodiment, the DNA of sudden change uses any suitable known mutagenesis procedures to include but not limited to use radiation, Asia The external generation such as nitroguanidine.It is then peeled off desired DNA sequence and in method provided herein.

As used herein, term " expands " and " gene amplification " refers to use and disproportionately replicates specific dna sequence Method so that the gene being amplified becomes to exist than the more high copy number that initially there are in genome.In some embodiments In, screen cell by growth in the presence of medicine (such as, the inhibitor of quenchable enzyme) and cause coding at medicine In the presence of grow the endogenous gene amplification of required gene outcome, or by (that is, the input of the external source of this gene outcome of amplification coding Thing) sequence or the two." amplification " is the special case of the nucleic acid replication including template specificity.It replicates with non-specific template (that is, be Template Dependent but do not rely on the duplication of specific template) is different.Template specificity is different from duplication and protects herein True property (that is, synthesizing correct polynucleotide sequence) and nucleotide (ribonucleotide or deoxyribonucleotide) specificity.Mould Plate specificity generally describes in " target " specific mode.Target sequence is that the implication of " target " is, they are sought from other The nucleic acid that nucleic acid sub-elects.Amplification technique primarily to such sub-electing and design.

As used herein, term " primer " refers to, the most naturally occurring, as in the restrictive diges-tion thing of purification or Synthetically produced, under conditions of being placed on the primer extension product that induction synthesis is complementary with nucleic acid chains (that is, at nucleotide and In the presence of derivant such as archaeal dna polymerase and at suitable temperature and pH) time can be as the synthesis few core that work of starting point Thuja acid.For the maximal efficiency expanded, primer is preferably strand, but double-strand alternatively.If double-strand, first locate Reason primer is to be used for before preparing extension products separating its chain.In some embodiments, primer is few deoxyribose core Thuja acid.Primer must long enough to cause the synthesis of extension products in the presence of derivant.As known in the art, primer Definite length will depend on many factors, including temperature, the source of primer and the use of method.

Term " mutagenic primer " and " mutagenic oligonucleotide " (being used interchangeably herein) refer to of corresponding templates sequence The oligonucleotide composition divided and can be hybrid with it.About mutagenic primer, this primer will not accurately match with template nucleic acid, One or more mispairing in primer are used to be incorporated in nucleic acid library desired sudden change.

As used herein, term " non-mutagenic primer " and " non-mutagenic oligonucleotide " (being used interchangeably herein) expection Refer to the oligonucleotide composition that will mate exactly with template nucleic acid.In some embodiments of the present invention, only employ and lure Become primer.In some other embodiments, primer is designed to so that at least one district including mutagenic primer Territory, also includes non-mutagenic primer in oligonucleotide mixture.By adding mutagenic primer and corresponding at least one mutagenic primer The mixture of non-mutagenic primer, it is possible to create present the obtained nucleic acid library of the mutant form of various combination.Such as, If it is desire to some members in mutant nucleic acid library retain its precursor sequence in some positions and other members in these sites are Sudden change, then non-mutagenic primer provides the specified level obtaining unmutated member for given residue in nucleic acid library Ability.The method of the present invention have employed the length between typically about 10-50 base, or it is highly preferred that about 15-45 base The mutation of length and non-mutagenic oligonucleotide.But, use that be shorter than about 10 bases or be longer than the primer of about 50 bases can Can be necessary to obtain desired mutagenesis results.About corresponding mutation and non-mutagenic primer, corresponding oligonucleotide has Identical length need not, but premise be in the region corresponding to sudden change to be added exist overlap.Can be with according to this Bright predetermined ratio adds primer.Such as, if it is desired to the library of gained has some specific sudden changes of significant level and less The different sudden changes of the identical or different site of amount, then the amount by adjusting the primer added produces desired skewed popularity library It is possible.Selectively, by adding less or more substantial non-mutagenic primer, it is right to produce in the nucleic acid library of adjustment sudden change The frequency that should suddenly change is possible.

As used herein, phrase " continuous mutation " refers to the sudden change being presented in same oligonucleotide primers.Such as, continuously Sudden change can be adjacent or be mutually adjacently, but, continuous mutation will be introduced in the mutagenesis template nucleic acid of gained by same primer.

As used herein, phrase " discontinuously sudden change " refers to the sudden change being presented in different oligonucleotide primers.Such as, Discontinuous sudden change will be introduced in the mutagenesis template nucleic acid of gained by the oligonucleotide primers prepared respectively.

As used herein, term " degenerate codon " refers to the codon for representing a different set of codon (also referred to as For " ambiguous codon ").Such as, degenerate codon " NNT " represent have 16 codons (A, C, T or G) of three base sequences/ The group of (A, C, T or G)/T.

As used herein, " coded sequence " refer to coded protein aminoacid sequence part polynucleotide (such as, Gene).

As used herein, term " probe " refers to, the most naturally occurring, as in the restrictive diges-tion thing of purification or Synthesis, restructuring or produced by PCR amplification, it is possible to the oligonucleotide of another targeted oligonucleotide acid hybridization (that is, a series of core Thuja acid).Probe can be strand or double-strand.Probe can be used on the detection of specific gene sequence, is identified and isolated from.Expection is originally Any probe used in invention will be with any " reporter molecules " labelling, thus it can detect in any detecting system, described Detecting system includes but not limited to enzyme (such as, ELISA and histochemical assays based on enzyme), fluorescence, radioactivity and sends out Photosystem.Do not expect that the present invention is limited to any specific detecting system or labelling.

As used herein, term " target ", when mentioning that polymerase chain reaction uses, refers to be used for polymerase chain reaction The nucleic acid region that the primer answered combines.Therefore, " target " is sought to sub-elect from other nucleotide sequences." section " is defined as target Nucleic acid region in sequence.

As used herein, term " polymerase chain reaction " (PCR) refers to U.S. Patent number 4,683,195,4,683,202 With 4, the method for 965,188, it is incorporated herein by reference, it includes target sequence in the mixture for increase genomic DNA The concentration of section and without clone or the method for purification.It is well known in the art for expanding this method of target sequence.

As used herein, term " amplifing reagent (amplification reagent) " refers to except primer, nucleic acid-templated Beyond amplification enzyme, those reagent (triphosphate deoxyribose nucleotide, buffer etc.) needed for amplification.Generally, amplifing reagent It is placed and is included in other reactive components in reaction vessel (test tube, micropore etc.).

As used herein, term " restriction endonuclease " and " limiting enzyme " refer to bacterial enzyme, its each comfortable particular core glycosides At acid sequence or neighbouring cutting double-stranded DNA.

" restriction site " refers to be designated restriction endonuclease identification and the nucleotide sequence of cracking, it is common that be used for Insert the site of DNA fragmentation.In some embodiments of the present invention, restriction site is by the engineered DNA construct of arriving In selected marker and 5' and 3' end.

As used herein, " homologous recombination " refers between two DNA moleculars or paired chromosome in identical or almost phase The site exchange DNA fragmentation of same nucleotide sequence.In some embodiments, chromosomal integration is homologous recombination.

As used herein, term " C1 " refers to thermophilic fungus destroyed wire, including described by Garg fungal bacterial strain (see, Garg, Mycopathol.,30:3-4[1966])。

As used herein, " Chrysosporium lucknowense " includes being described in U.S. Patent number 6,015,707, 5,811,381 and 6,573,086；U.S. Patent Publication number 2007/0238155, US2008/0194005, US2009/ 0099079；International Patent Publication No. W WO2008/073914 and the bacterial strain of WO98/15633, it is all incorporated herein by, And include but not limited to Chrysosporium lucknowense Garg27K, VKM-F3500D (registration number VKM F- 3500-D), C1 bacterial strain UV13-6 (registration number VKM F-3632D), C1 bacterial strain NG7C-19 (registration number VKM F-3633D) and C1 Bacterial strain UV18-25 (VKM F-3631D), it is all deposited in Russian Academy Of Sciences full Russia Culture Collection (VKM), Bakhurhina St.8, Moscow, Russia, 113184, and its any derivative strain (derivative).To the greatest extent Pipe is originally described as Chrysosporium lucknowense, is presently thought that C1 is the bacterial strain of thermophilic fungus destroyed wire.Other C1 Bacterial strain include with registration number ATCC44006, CBS (Centraalbureau voor Schimmelcultures) 122188, The cell of CBS251.72, CBS143.77, CBS272.77, CBS122190, CBS122189 and VKM F-3500D preservation.Example Property C1 derivative strain includes that wherein one or more endogenous genes or sequence are lacked or modify and/or one or more external source base The organism of the modification that cause or sequence are introduced into.Derivative strain includes but not limited to UV18#100f Δ alpl, UV18#100f Δ Pyr5 Δ alp1, UV18#100.f Δ alp1 Δ pep4 Δ alp2, UV18#100.f Δ pyr5 Δ alp1 Δ pep4 Δ alp2 and UV18#100.f Δ pyr4 Δ pyr5 Δ aIp1 Δ pep4 Δ alp2, as described by WO2008073914 and WO2010107303 , its each piece is incorporated herein by.

As used herein, term " is cultivated " and is referred to grow in liquid or solid culture medium under suitable condition microorganism The colony of cell.Expection is cultivated in any suitable form, equipment (such as, shaking flasks, fermentation tank, bioreactor etc.) enters OK.Use any suitable process to carry out it is also contemplated that cultivate, include but not limited in batches, batch feeding and/or continuously Cultivate.It is true that expect that any combination of suitable method can use.

In " batch process ", when operation starts, in addition to the oxygen for aerobic technique, by all necessary materials Place in the reactor and allow fermentation to carry out until completing, now gathering in the crops product.In some embodiments, it is used for producing this The batch process of fungal cell, enzyme and/or the enzymatic mixture of invention is carried out in shaking flasks or bioreactor.

In " batch feeding technique ", culture is supplied continuously or sequentially one or more nutrient media componentses, and not Remove and cultivate fluid.

In " continuous processing ", supply fresh culture continuously with the ratio that volume is equal and remove cultivation fluid, with Keep culture in stable growth rate.When mentioning continuous processing, " stable state " refers to the inconspicuous change of concentration of wherein reactant State, " quasi-stable state " refers to wherein after reaction starts, and the concentration of reactant is in the normal operating one with continuous hydrolysis technology The state of fluctuation in the scope caused.

As used herein, term " saccharifying " refers to that its mesostroma (such as, cellulose biomass) is by the effect of cellulase It is broken to produce the process of fermentable saccharide (such as, monosaccharide, such as, but not limited to glucose).

As used herein, term " fermentable saccharide " refers to simple saccharide (such as, monosaccharide, disaccharide and little oligosaccharide), Include but not limited to glucose, xylose, galactose, arabinose, mannose and sucrose.It is true that fermentable saccharide is micro-life Any sugar that thing can utilize or ferment.

As used herein, term " soluble saccharide " refers to water solublity pentose and hexose monomer and most about 6 monomer lists The oligomer of unit.Expect that any water solublity monosaccharide and/or oligosaccharide contained in this term.

As used herein, term " ferments " and broadly uses, and refers to the oxygen from organic compound (such as, carbohydrate) Change the process obtaining energy.It is true that " fermentation " broadly refers to that carrying out chemical conversion sugar source via use fermenting organisms is End-product.In some embodiments, this term contain utilize saccharide such as fermentable saccharide as the energy obtain expectation produce The microorganism of thing or the cultivation of culture of microorganism.

As used herein, term " fermenting organisms " refers to be suitable to produce any organism of desired end-product, including Protokaryon and most eukaryotes (such as, bacterial organisms and fungal organism such as yeast and filamentous fungi).Especially suitable Fermenting organisms can ferment directly or indirectly (that is, convert) saccharide, include but not limited to glucose, fructose, maltose, Xylose, mannose and/or arabinose are at least one desired end-product.In some embodiments, can be used for the present invention Yeast include but not limited to belong to Saccharomyces (Saccharomyces) bacterial strain (such as, saccharomyces cerevisiae (Saccharomyces Cerevisiae) and the bacterial strain of Saccharomyces uvarum (Saccharomyces uvarum)), belong to pichia (Pichia) bacterium Strain (such as, pichia stipitis (Pichia stipitis) such as pichia stipitis CBS5773 and pichia pastoris phaff (Pichia pastoris)) and bacterial strain (such as, the Candida utilis (Candida of genus mycocandida (Candida) Utilis), Candida arabinofermentans, Di Dansi candida mycoderma (Candida diddensii), Candida Sonorensis, shehatae candida (Candida shehatae), candida tropicalis (Candida tropicalis) With Candida boidinii (Candida boidinii)).Other fermenting organisms include but not limited to zymomonas (Zymomonas), Hansenula (Hansenula) (such as, multiple-shaped nuohan inferior yeast (Hansenula polymorpha) and different Often Hansenula yeast (Hansenula anomala)), Kluyveromyces (Kluyveromyces) (such as, crisp wall Crewe dimension ferment Female (Kluyveromyces fragilis)) and Schizosaccharomyces (Schizosaccharomyces) (such as, foxtail millet wine fragmentation ferment Female (Schizosaccharomyces pombe)) bacterial strain.

As used herein, term " slurry (slurry) " refers to be dispersed with one or more solid constituents wherein, such as fine The aqueous solution of dimension element substrate.Therefore, the suspension referring to solid in a liquid " starched " in term.In some embodiments, cellulose Substrate is in a liquid with concentration pulp that is thick but still that can be pumped.Such as, in some embodiments, liquid is water, returns Skill of knocking off stream and/or the effluent processed.But, do not expect that the present invention is limited to any particular liquid and/or solid.

Term " biomass " and " biomass substrate " contain any suitable material in saccharification react.This term is contained Lid but be not limited to, including cellulose (that is, " cellulose biomass ", " cellulosic material " and " cellulose matrix ") and wooden The material of cellulose biomass.It is true that term " biomass " any work of including comprising polysaccharide matrix or the biology of death Material, includes but not limited to cellulose, starch, the long chain carbohydrates polymer of other forms and the mixing in these sources Thing.In some embodiments, it entirely or predominately assembles from glucose or xylose, and in some embodiments, appoints Selection of land also comprise multiple other pentose and/or hexose monomer.Biomass can be obtained from plant, animal or microorganism, and includes But it is not limited to be obtained from plant, animal or microorganism, and includes but not limited to agricultural, industry and forestry residue, industry and city Political affairs garbage and the land planted for energy purpose and aquatic crops.The example of biomass substrate includes but not limited to, Wood, wood pulp, paper pulp, corn fiber, niblet, corn cob, corn residue such as skin of Semen Maydis, corn straw, grass, Semen Tritici aestivi, wheat Stalk, Fructus Hordei Vulgaris, barley-straw, Radix Glycyrrhizae, rice, rice straw, switchgrass, waste paper, paper and paper pulp processing waste material, wooden or herbaceous plant, pulp Or vegetable, distiller grains, grass, rice husk, Cotton Gossypii, Fructus Cannabis, Caulis et Folium Lini, Folium Agaves Sisalanae, bagasse, Sorghum vulgare Pers., Semen sojae atricolor, switchgrass, corn of milling obtain Component, tree, branch, root, leaf, wood chip, sawdust, bushes, vegetable, fruit and flower and its any suitable mixture. In some embodiments, biomass include but not limited to raise crop (such as, grass, include C4 grass, such as switchgrass, rice Grass, rye grass, Miscanthus, reed canary grass or its any combination), sugar process residues, such as but not limited to, bagasse (such as, bagasse, sweet Puree [such as sugar beet] or a combination thereof), agriculture residues (such as, soybean stalk, corn straw, corn fiber, rice straw, sweet Sugarcane stalk, rice, rice husk, barley-straw, corn cob, straw, Rape Straw, Herba bromi japonici stalk, oat shell, corn fiber, Fructus Cannabis, Caulis et Folium Lini, Folium Agaves Sisalanae, Cotton Gossypii or its any combination), pulp, vegetable, distiller grains, forestry biomass (such as, the wood pulp of wood, wood pulp, paper pulp, recovery Fiber, sawdust, hardwood such as poplar, soft wood or a combination thereof).And, in some embodiments, biomass include fibre Dimension element waste material and/or forest trimmings, includes but not limited to, paper and paper pulp processing waste material, city paper waste material, newsprint, cardboard and Analog.In some embodiments, biomass include a type of fiber, and in some alternate embodiment, raw Material includes the mixture deriving from the fiber of different biomass.In some embodiments, biomass also include expressing wooden Element enzyme and/or the transgenic plant (see for example, US2008/0104724A1) of cellulase.

As used herein, " lignocellulose " refers to the substrate (matrix) of cellulose, hemicellulose and lignin.From wood Matter cellulose biomass produce economically bio-fuel generally include conversion cellulose and hemi-cellulose components be fermentable saccharide, It is typically monosaccharide such as glucose (from cellulose) and xylose and arabinose (from hemicellulose).Convert the most completely and can lead to Cross Chemical Pretreatment lignocellulose, realize with the hydrolysis of cellulase enzymatic subsequently.Chemical pretreatment step makes cellulose More sensitive to enzymatic hydrolysis, and in some cases, also hydrolyzed hemi-cellulose components.Multiple Chemical Pretreatment technique is this Known to field, include but not limited to, in weak acid (mild acid) pretreatment and diluted acid, the ammonium pretreatment or organic molten of high temperature Agent is extracted.

Lignin is more more complicated than cellulose or hemicellulose and heterogeneous biopolymer, single including multiple phenolic aldehyde Asia Position (phenolic subunit).Enzymatic lignin depolymerization can be by the lignin peroxidase of usual co-action, manganese mistake Oxide enzyme, laccase and cellobiose dehydrogenase (CDH) complete.But, as title represents, CDH enzyme also oxidized fibre two Sugar is cellobiose acid lactone.Many report expressions, owing to reducing the concentration of cellobiose, are strengthened by CDH oxidized fibre disaccharide The ratio of cellulase hydrolysis cellulose, cellobiose is the potent inhibitor (Mansfield etc. of some fibre element enzyme component People, Appl.Environ.Microbiol., 63:3804-3809 [1997]；With Igarishi et al., Eur.J.Biochem., 253:101-106[1998]).The most it be reported that, the cellulose decomposition that CDH can strengthen from glycosyl hydrolase family 61 strengthens The activity (see for example, WO2010/080532A1) of albumen (cellulolytic enhancing protein).

As used herein, term " lignocellulose biomass " refers to cellulose and half fiber being combined with lignin Any plant biomass of element.

In some embodiments, optionally by chemistry, physics and Biological Pretreatment (such as steam explosion, slurrying, stone roller Mill, acid hydrolysis, solvent exposure etc., with and combinations thereof) preprocessing biomass with increase cellulose to hydrolysis sensitivity.Multiple Lignocellulosic material can use, including comprising fresh lignocellulosic material, partially dried lignocellulosic material, completely Be dried lignocellulosic material and/or its any combination of those.In some embodiments, lignocellulosic material bag Include more than about 20%, be more preferably more than about 30%, be more preferably more than the cellulose of the amount of about 40% (w/w).Such as, at some In embodiment, ligno-cellulosic materials includes cellulose or any amount therebetween of from about 20% to about 90% (w/w), and In some embodiments, ligno-cellulosic materials includes less than about 19%, less than about 18%, less than about 17%, less than about 16%, be less than About 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, few In about 7%, less than about 6% or less than the cellulose (w/w) of about 5%.And, in some embodiments, lignocellulosic material Including the amount more than about 10%, the lignin of the amount being more generally more than about 15% (w/w).In some embodiments, wooden fibre Dimension element raw material includes a small amount of sucrose, fructose and/or starch.Lignocellulosic material generally first pass through include but not limited to Under method stand size reduce: mill, grind, vibrate, tear up, suppress/expand or other kinds of mechanism.Pass through The size of mechanism reduces and can carry out by adapting to any kind of equipment of this purpose, such as but not limited to, hammer type crushing Machine, drum-type crusher, roll press, refiner and hydrabrusher.In some embodiments, generation is reduced from size By weight at least the 90% of granule has the length of less than about 1/16 to about 4 inch (in), and (measured value can be volume or weight Average length).In some embodiments, the equipment being used for reducing particle diameter is beater grinder or shredder.Reduce in size After, generally by raw material pulp in water, because this promotes the pumping of raw material.In some embodiments, particle diameter less than about 6 inches Lignocellulosic material need not size reduce.

As used herein, term " lignocellulosic material " refers to any type of the raw material being suitable for use as in saccharification react Lignocellulose biomass.

As used herein, term " lignocellulosic material of pretreatment " refers to as it has been described above, by physics and/or change Learn technique so that fiber more and and/or can more be easily accepted by the lignocellulosic material of cellulolytic enzyme effect.

As used herein, term " lignocellulose-competence (lignocellulose-competent) ", " wooden fibre Dimension element-utilize (lignocellulose-utilizing) " and similar terms refer to secretion participate in lignin destroy and hydrolyze The organism of enzyme.Such as, in some embodiments, it is wooden that lignocellulose-competence fungal cell secretes one or more Element peroxidase, manganese peroxidase, laccase and/or cellobiose dehydrogenase (CDH).Required these of lignin degradation Exoenzyme is commonly referred to as " lignin modification enzyme (lignin-modifying enzyme) " or " LME ".

When biomass substrate by some physically and/or chemically means processing be easy to saccharifying time, this biomass substrate It is referred to as " pretreatment ".As further described herein, in some embodiments, use methods known in the art " pre- Process " or process biomass substrate, such as Chemical Pretreatment (such as, ammonia pretreatment, dilute acid pretreatment, diluted alkaline pretreatment or molten Agent exposes), physics pretreatment (such as, steam explosion or irradiation), mechanical pretreatment (such as, grind or mill) and biology locate in advance Reason (such as, apply the microorganism of solubilising lignin) and a combination thereof, to increase the cellulose sensitivity to hydrolysis.

In some embodiments, before pre-processing by substrate pulp.In some embodiments, the concordance of slurry It is about 2% to about 30%, the most about 4% to about 15%.In some embodiments, before pre-processing slurry is carried out water And/or acid soak operation.In some embodiments, use any applicable method by de-watering of slurries to subtract before pre-processing Few steam and the use of chemicals.The example of dehydration device includes but not limited to pressing screw press (pressurized Screw press) (see for example, WO2010/022511, be incorporated herein by) pressure filter and extruder.

In some embodiments, pretreatment is carried out with half fiber present in hemicellulose and/or lignocellulose Element partial hydrolysis is monomer pentose and hexose saccharide (such as, xylose, arabinose, mannose, galactose and/or its any group Close).In some embodiments, carry out pretreatment thus occur the almost complete hydrolysis of hemicellulose and cellulose to glucose A small amount of conversion.In some embodiments, from about 0.02% (w/w) to about 2% (w/w) or therebetween any in aqueous slurry The acid concentration of amount is generally used for processing cellulose matrix.Any applicable acid can be used for these methods, includes but not limited to, salt Acid, nitric acid and/or sulphuric acid.In some embodiments, the acid used during pretreatment is sulphuric acid.Steam explosion is by raw A kind of method (see for example, U.S. Patent number 4,461,648) of the low-kappa number of material matrix.The another kind of pretreatment slurry Method includes continuous pretreatment (that is, cellulose biomass pumping into reactor continuously).This method is people in the art Member is known (see for example, U.S. Patent number 7,754,457).

In some embodiments, pretreatment uses alkali.Being different from low-kappa number, oxygenation pretreatment can not hydrolyze biology The hemi-cellulose components of matter.But, acidic-group present on alkali and hemicellulose reacts the surface opening substrate.At some In embodiment, the crystal structure adding change cellulose of alkali, so that cellulose is more in compliance with hydrolysis.Can be used for pretreatment The example of alkali include but not limited to, ammonia, ammonium hydroxide, potassium hydroxide and sodium hydroxide.A kind of method of oxygenation pretreatment is ammonia Freezing blasting procedure, ammonia fiber blasting procedure or ammonia filament expansion method (" AFEX " technique；See for example, U.S. Patent number 5,171, 592；5,037,663；4,600,590；6,106,888；4,356,196；5,939,544；6,176,176；5,037,663 and 5, 171,592).During this technique, in pressurizing vessel, cellulose matrix is contacted time enough with ammonia or ammonium hydroxide So that ammonia or ammonium hydroxide can change the crystal structure of cellulose fibre.Reducing pressure the most rapidly, this allows ammonia flash distillation Or boiling also explosion cellulosic fibrous structure.In some embodiments, methods known in the art are then used to reclaim flash distillation Ammonia.In some alternative methods, use dilute ammonia pretreatment.Dilute ammonia pretreatment method utilizes the ammonia diluter than AFEX or hydroxide Ammonium salt solution (see for example, WO2009/045651 and US2007/0031953).This pretreating process can produce or not produce appoints What monosaccharide.

Other pretreating process used in this invention includes, with organic solvent chemical treatment cellulose matrix, such as existing Pretreatment system utilizes those methods (see for example, U.S. Patent number 4,556,430) of organic liquid.These methods have Following benefit: low-boiling point liquid can easily recycle.Other pretreatment, such as Organosolv^TMTechnique, also makes With organic liquid (see for example, U.S. Patent number 7,465,791).Making substrate stand pressure (hydraulic) water can also be applicable pretreatment Method (see for example, Weil et al., Appl.Biochem.Biotechnol., 68:21-40 [1997]).Some embodiment party In case, after pre-processing, by the cellulose biomass of any one processing pretreatment of multiple steps, described step such as water Diluting, wash, buffer, filter or centrifugal or any combination of these techniques, enzymatic hydrolysis subsequently, such as those skilled in the art It is familiar with.

Pretreatment produces and comprises the solvable of the saccharide, optionally acetic acid and other inhibitor that include hydrolysis of hemicellulose and produce Component and include feedstock composition (the such as, " raw material of pretreatment of pretreatment of solid of unhydrolysed raw material and lignin Slurry ").In some embodiments, from the soluble constituent of solid separation and culture feedstock composition to produce solvable fraction.? In some embodiments, then can by include the saccharide of release during pretreatment and other soluble constituents (such as, inhibitor) Molten fraction sends to fermentation.But, in some embodiments that hemicellulose is not effectively hydrolyzed during pre-processing wherein, Including one or more other steps (such as, further hydrolysing step and/or enzymatic treatment step and/or further Alkali and/or acid treatment) to produce fermentable saccharide.In some embodiments, by the raw material with solution washing pretreatment Compositions separates, to produce the efflux of solids of washing liquid stream and the raw material that comprises unhydrolysed, pretreatment.Alternatively, pass through (such as, centrifugal, microfiltration, filter press, cross-flow filtration, pressure filtration, vacuum filter to use any applicable method Deng) feedstock composition of pretreatment is carried out solid-liquid separation, from solid separation soluble constituent.Optionally, in some embodiments In, solid-liquid separation mixes washing step.In some embodiments, then to comprising the solid of separation of cellulose with fibre Dimension element enzyme carries out enzymatic hydrolysis to convert cellulose into glucose.In some embodiments, by the material combination of pretreatment Thing is fed into fermentation technology and does not separate the solid that wherein comprises.In some embodiments, to non-water after fermentation technology The solid solved carries out enzymatic hydrolysis to convert cellulose into glucose with cellulase.In some embodiments, to pre-place The cellulosic material of reason carries out enzymatic hydrolysis with cellulase.

As used herein, term " chemical treatment " refer to promote cellulose, hemicellulose and/or the separation of lignin and/ Or any Chemical Pretreatment of release.

As used herein, term " physics pretreatment " refers to promote that cellulose, hemicellulose and/or lignin are from cellulose The separation of material and/or any pretreatment of release.

As used herein, term " mechanical pretreatment " refer to for process biomass any machinery means, including but not It is limited to polytype grinding or mill (such as, dry grinding, wet grinding or vibratory milling).

As used herein, term " Biological Pretreatment " refers to promote that cellulose, hemicellulose and/or lignin are from cellulose The separation of material and/or any Biological Pretreatment of release.

As used herein, term " reclaims " and refers to gather in the crops from cell and/or culture medium, separate, collect or reclaim albumen.? In the background of saccharifying, use and reclaim the fermentable saccharide referring to produce during saccharification react from culture medium and/or cell harvesting. In the background of fermentation, recovery is used to refer to from culture medium and/or cell harvesting tunning.Therefore, if a process is wrapped Include after at least some product is generated in the reaction, from other Component seperation product of reactant mixture, then should Process is referred to as including " recovery " product (soluble sugar such as reclaimed) from saccharifying.

As used herein, when reaction exists target specific components (such as, enzyme) and under the same conditions with same base Matter and other subs are carried out, but the reaction that there is not target components (such as, there is not enzyme) is compared, and results in more product Time, occur " increasing " to carry out the yield of self-reacting product (such as fermentable saccharide).

As used herein, if compared with other enzymes participating in catalytic reaction, the amount of certain enzyme is less than about 2%, about 1% or about 0.1% (wt/wt), then reaction is referred to as the specific enzyme of substantially free.

As used herein, " classification " liquid (such as, culture broth) refers to apply separating technology (such as, salt precipitation, post color Spectrum, size exclusion and filtration) or this type of technique be combined to provide a kind of solution, wherein expect albumen (such as, cellulase, And/or a combination thereof) than the bigger percentage ratio constituting total protein in initial liquid product.

As used herein, term " enzymatic hydrolysis " refers to by enzyme hydrolysis substrate.In some embodiments, hydrolysis includes it The middle method at least one enzyme contacted with at least one substrate to produce end-product.In some embodiments, enzymatic hydrolysis Method includes at least one cellulase and acts at least one glycosidase and/or the mixture of polysaccharide (such as, cellulose) Glycosidase, to be completely or partially converted into fermentable saccharide by it.When glycosidic bond between two monosaccharide present in substrate At least some is hydrolyzed, thus by time separated from one another for two monomers connecting before this, " the hydrolysis of cellulose or other polysaccharide occurs " and/or " hydrolysis (hydrolysis) " (hydrolyzing).

Expection enzymatic hydrolysis is can hydrolyze any suitable type that at least one substrate is at least one end-product Enzyme is carried out.In some embodiments, substrate is cellulose, and in some other embodiments, substrate is lignocellulose, In embodiment further, substrate is another kind of compositions (such as, starch).In some embodiments, end-product Including at least one fermentable saccharide.It is also contemplated that enzymatic hydrolysis contain with can hydrocellulose as glucose any suitably The process that carries out of the cellulase of type, regardless of whether the source of cellulase.Expect that the enzyme in any suitable source can be used for The present invention, includes but not limited to from fungus, such as trichoderma (Trichoderma spp.), aspergillus (Aspergillus Spp.), Hypocrea (Hypocrea spp.), Humicola (Humicola spp.), neurospora (Neurospora Spp.), Orpinomyces spp., gibberella belong to (Gibberella spp.), Emericella (Emericella spp.), hair Shell belongs to (Chaetomium spp.), Chrysosporium (Chrysosporium spp.), Fusarium (Fusarium spp.), penicillium sp Belong to seat shell between (Penicillium spp.), big angle and belong to (Magnaporthe spp.), raw wool flat lead fungi genus (Phanerochaete spp.), Trametes (Trametes spp.), Lentinus Edodes (Lentinula edodes), Gleophyllum trabeiu、Ophiostoma piliferum、Corpinus cinereus、Geomyces pannorum、Cryptococcus laurentii、Aureobasidium pullulans、Amorphotheca resinae、Leucosporidium scotti、 Cunninghamella elegans, Thermomyces lanuginosus, Myceliopthora thermophila and addicted to The enzyme that hot side spore is mould, and subordinate bacillus (Bacillus), Thermomyces, fusobacterium (Clostridium), chain Those of the antibacterial acquisition of mould Pseudomonas (Streptomyces) and Thermobifida.

In some embodiments, enzymatic hydrolysis for or enter close to the pH of optimum and temperature using cellulase OK.Such as, in some embodiments, enzymatic hydrolysis is about 30 DEG C to about 75 DEG C or any suitable temperature therebetween, such as About 30 DEG C, about 35 DEG C, about 40 DEG C, about 45 DEG C, about 50 DEG C, about 55 DEG C, about 60 DEG C, about 65 DEG C, about 70 DEG C, the temperature of about 75 DEG C or Any temperature therebetween and the pH of about 3.5 to about 7.5 or any pH therebetween (such as, about 3.5, about 4.0, about 4.5, about 5.0, About 5.5, about 6.0, about 6.5, about 7.0, about 7.5 or any suitable pH therebetween) carry out.In some embodiments, enzymatic Hydrolysis starts initial concentration preferably about 0.1% (w/w) to about 20% (w/w) or any suitable amount therebetween of front cellulose (such as, about 0.1%, about 0.5%, about 1%, about 2%, about 4%, about 6%, about 8%, about 10%, about 12%, about 14%, about 15%, about 18%, about 20% or any suitable amount therebetween).In some embodiments, the unitized dose of all fibres element enzyme is every gram of cellulose About 0.001 to about 100mg albumen or any suitable amount therebetween (such as, every gram of cellulose about 0.001, about 0.01, about 0.1, About 1, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100mg albumen or Any amount therebetween).Enzymatic hydrolysis carries out any reasonable time section.In some embodiments, enzymatic hydrolysis carries out about 0.5 Hour to the time period of about 200 hours or any time therebetween (such as, about 2 hours to about 100 hours or therebetween any suitable The conjunction time).Such as, in some embodiments, its carry out about 0.5, about 1, about 2, about 5, about 7, about 10, about 12, about 14, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 120, about 140, about 160, about 180, about 200 hours or any applicable time therebetween.

In some embodiments, enzymatic hydrolysis is batch hydrolysis, continuous hydrolysis and/or a combination thereof.Some embodiment party In case, hydrolysis is that be stirred, unmixing or a combination thereof.Enzymatic hydrolysis is generally carried out in hydrolysis reactor.Pre-adding Cellulase composition, to before, during or after hydrolysis reactor, is added pretreatment by the lignocellulose substrate processed Lignocellulose substrate.It is true that not anticipation reaction condition be limited to provided herein those because amendment fully in this area In technical staff's ken.In some embodiments, after cellulose hydrolyzes, before any being processed further, use Any insoluble solid present in the ligno-cellulose hydrolysate of Conventional solid-liquid separation technology removing gained, including But it is not limited to lignin.In some embodiments, burn these solids think whole technique provide energy.

As used herein, " available total fiber element " be enzymatic hydrolysis can and the amount (wt%) of cellulose.Available total fibre The amount of the cellulose that dimension element is generally equal to or closely initial equal to present in hydrolysis.

As used herein, " residual fiber element " is that in hydrolysed mix, available total fiber element keeps unhydrolysed ratio (wt%).Residual fiber element can use any suitable method known in the art to measure.Such as, IR spectroscopy can be used direct The amount of the glucose measuring the Concentrated acid hydrolysis generation that maybe can be determined by remaining solid is measured.

As used herein, " the total fiber element of hydrolysis " is the ratio of the available total fiber element of hydrolysis in hydrolysed mix. Such as, the total fiber element of hydrolysis can be calculated as the difference of " available total fiber element " and " residual fiber element ".

As used herein, " theoretical maximum glucose yield " is can to produce from available total fiber element under specified requirements The maximum (wt%) of glucose.

As used herein, " Gmax " refers to the maximum (wt%) of the glucose that the total fiber element from hydrolysis produces. Gmax can be calculated as below: such as, is specifying under reaction condition remaining residual fiber element at the end of reaction by directly measuring Amount, from available total fiber element deduct residual fiber element amount with determine hydrolysis total fiber element, then calculate can be from water The amount of the glucose that the total fiber element solved produces.

It will be appreciated by those skilled in the art that when computational theory value such as Gmax and theoretical maximum glucose yield, The quality of two hydrogen atoms and an oxygen atom that join glucose molecule in hydrolysis reaction is taken into account.Example As, when the polymer of " n " glucose unit is hydrolyzed, the water of (n-1) unit is added in hydrolysis the glucose molecule formed, So the weight about 10% of the cellulose consumed in the weight ratio hydrolysis of the glucose produced (such as, hydrolyzes 1g cellulose will produce Raw about 1.1g glucose).

Accordingly, as an example, there is 5g available total fiber element when hydrolysis starts, and after reaction, remain 2g During residual fiber element, the total fiber element of hydrolysis is 3g cellulose.The theoretical maximum glucose of 100% (w/w) at reaction conditions Yield is about 5.5g glucose.Gmax is based on being hydrolyzed release or the 3g cellulose calculating converted in reaction.Therefore, at this In example, the Gmax of 100% (w/w) is about 3.3g glucose.Cellulose Levels, either available total amount present in substrate Or unhydrolysed or that residual fiber is plain amount, can quantitatively, such as to pass through by any one of multiple method known in the art IR spectroscopy or by measure cellulose Concentrated acid hydrolysis produce glucose amount (see for example, U.S. Patent number 6,090, 595 and 7,419,809).

As used herein, term " insoluble solids " refers to be suspended in liquid but insoluble solid material.As this area is public Know, suspend or the concentration of insoluble solids can be determined by any suitable method (such as, by using glass microfibre filter paper Filter the sample of slurry, wash filter cake with water, and at about 105 DEG C of dry cakes overnight).

As used herein, term " unhydrolysed solid ", " unconverted solid " and similar terms refer to not by cellulose Present in the cellulose of enzymic digestion and raw material, non-cellulosic materials inert to cellulase or other materials.

As used herein, term " by-product " refers to organic point of special process (such as, saccharifying) less desirable product Son.

As used herein, term " antibody " refers to immunoglobulin.Antibody includes but not limited to obtain anti-from it from expectation The immunoglobulin that any species of body directly obtain.It addition, the present invention includes the antibody modified.This term also refers to that reservation finishes Close the antibody fragment of the ability of the epi-position that complete antibody combines, and include polyclonal antibody, monoclonal antibody, chimeric antibody, resist Idiotype (anti-ID) antibody.Antibody fragment includes but not limited to complementary determining region (CDR), single-chain fragment variable region (scFv), weight Chain variable region (VH) and variable region of light chain (VL) fragment.

As used herein, term " the most stable " and " heat-staple " refer to when being exposed to the temperature of change, Use main during enzyme under conditions of be exposed to the time period that the temperature determined is given after remain the enzymatic activity of specified quantitative The enzyme of the present invention." temperature of change " includes the temperature being raised and lowered.In some embodiments, these enzymes change being exposed to The temperature specific time period become, such as, at least about 60 minutes, about 120 minutes, about 180 minutes, about 240 minutes, about 300 points Remain at least about 50% after clock etc., about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98% or the enzymatic activity of about 99%.

As used herein, term " thermophilic fungal " refers at least about 37 DEG C, and usually less than about 100 DEG C, the most such as Between about 37 DEG C to about 80 DEG C, between about 37 DEG C to about 75 DEG C, between 40 DEG C to about 65 DEG C, or about 40 DEG C to about 60 DEG C it Any fungus of optimum growh is demonstrated at a temperature of between.Generally, optimum growh shows the temperature at least about 40 DEG C to about 60 DEG C Under degree.

As used herein, " solvent stability " refers to compared with reference polypeptide, is exposed to variable concentrations (such as, about 5% To about 99%) nonaqueous solvent (such as, isopropanol, oxolane, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, first Base tertbutyl ether etc.) keep after a period of time (such as, about 0.5 to about 24 hour) shares activity (such as, greater than about 60% to About 80%) polypeptide.

As used herein, when term " oxidation-stabilized " refers to such as be exposed to oxidant or catalytic oxidation agent, using During the present invention main under conditions of retain the enzyme of the present invention of enzymatic activity of specified quantitative through the given time period.Implement at some In scheme, these enzymes are in the time period specific with oxidising agent, such as, at least about 1 minute, about 3 minutes, about 5 minutes, about 8 Minute, about 12 minutes, about 16 minutes, remain at least about 50% after about 20 minutes etc., about 60%, about 70%, about 75%, about 80%, About 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98% or the enzymatic activity of about 99%.

As used herein, " pH is stable " refers to compared with reference polypeptide, be exposed to low or high pH (such as, about 4.5 to About 6 or about 8 to about 12) (such as, greater than about 60% to about to keep shares activity after a period of time (such as, 0.5-24 hour) 80%) polypeptide.

As used herein, the term " stability of enhancing " under the enzyme background that oxidation, chelating agen, heat and/or pH are stable Refer to elapse in time the enzymatic activity of higher reservation compared with other enzymes and/or wild-type enzyme.

As used herein, the term " stability of minimizing " under the enzyme background that oxidation, chelating agen, heat and/or pH are stable Refer to compared with other enzymes and/or wild-type enzyme, elapse the enzymatic activity of lower reservation in time.

Detailed Description Of The Invention

The invention provides the fungal bacterial strain of improvement.In some embodiments, the fungal bacterial strain of described improvement can be used for Cellulosic material hydrolysis is become glucose.As noted herein, the invention provides have minimizing endogenous cellobiose take off The secreting active fungal bacterial strain of hydrogen enzyme.In some embodiments, fungal bacterial strain secretion improves fermenting from cellulose The enzymatic mixture of candy output.Report before has shown that cellobiose dehydrogenase enhances cellulose to the oxidation of cellobiose The speed of enzyme hydrolysis cellulose.Different from the conventional wisdom in this area, the present invention provides minimizing fiber two astoundingly The activity of glucocorticoid dehydrogenase also causes the genomic modification of output increased of the fermentable sugars from cellulose.Advantageously, carry herein The fungal cell's secretion producing cellulase of the genetic modification of confession causes the such as Portugal of the fermentable sugars from cellulose improved The enzymatic mixture of the yield of grape sugar.

Lignocellulose (also referred to as " lignocellulose biomass ") comprises the base of cellulose, hemicellulose and lignin Matter.Produce bio-fuel from lignocellulose biomass economically and generally include that cellulose and hemi-cellulose components are changed into can The sugar of fermentation, it is common that monosaccharide such as glucose (from cellulose) and xylose and arabinose (from hemicellulose).Several Convert completely and can pass through Chemical Pretreatment lignocellulose, next realize with the hydrolysis of cellulase enzymatic.The pre-place of chemistry Reason step makes cellulose more susceptible to enzymatic hydrolysis, and in some cases, goes back hydrolyzed hemicellulose component.This area is The many Chemical Pretreatment techniques known can with in the present invention, and include but not limited to the weak acid pretreatment under high temperature and diluted acid, Ammonium pretreatment and/or organic solvent extraction.

Cellulase is typically to play synergistically cellulose decomposition becomes solubility disaccharide or oligosaccharide such as cellobiose The different types of cellulolytic enzyme of effect (such as, endoglucanase and cellobiohydrolase, the latter is also referred to as " exoglucanase ") mixture, then cellobiose is further hydrolyzed to glucose by β-glucosyl enzym.Cellulase by Various microorganisms produce.From the cellulase of filamentous fungi and some antibacterials and hemicellulase by extensive land productivity In the many commercial Application relating to processing natural fiber saccharogenesis.

Lignin is the biopolymer more complicated and more heterogeneous than cellulose or hemicellulose and includes that multiple phenol Asia is single Position.Enzymatic lignin depolymerization can be by the lignin peroxidase generally acted on synergistically, manganese peroxidase, laccase And/or cellobiose dehydrogenase (CDH) realizes.But, as the name implies, cellobiose is also oxidized to fiber by CDH enzyme Two sugar lactones.Multiple report shows, cellobiose is aoxidized and enhances fiber owing to decreasing the concentration of cellobiose by CDH The speed of element enzyme hydrolysis cellulose, cellobiose is that the potent inhibitor of some fibre element enzyme component (see for example Mansfield Et al., Appl.Environ.Microbiol., 63:3804-3809 [1997]；With Igarishi et al., Eur.J.Biochem.,253:101-106[1998]).Recently, reported that CDH can strengthen from glycosyl hydrolase family 61 Cellulolytic strengthens the activity (see for example, WO2010/080532A1) of albumen.

Among the filamentous fungi of cellulase-producing, exist and also produce those of multiple enzyme participating in lignin degradation.Example As, such as belong to myceliophthora, Chrysosporium, Sporotrichum, Thielavia, the flat lead fungi of raw wool belong to and Trametes organism produce and Eccrine fiber element enzyme, hemicellulase and the mixture of lignin-degrading enzymes.The organism of these types is wooden due to its digestion The ability of element and be commonly called " white rot fungi ", be different from " palm fibre rotten " fungus (the such as Trichoderma spp. being generally not capable of digesting lignin Belong to).

The fungal cell of genetic modification

The fungal cell of genetic modification provided herein allows to reduce and is taken off by the endogenous cellobiose of described emiocytosis The amount of hydrogenase activity.In the fungal cell of genetic modifications more provided herein, the cellobiose of described cell separation take off The activity of hydrogen enzyme is secreted relative to by the parent fungal cell of the unmodified grown under essentially identical condition of culture or cultivate The activity level of cellobiose dehydrogenase reduce at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, About 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more.In some embodiments, genetic modification is true Bacterium cell provides relative to by the parent fungal cell secretion of the unmodified grown under substantially the same conditions or cultivate At least about the 5% of the activity level of cellobiose dehydrogenase, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, About 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more.

Will be apparent from, any suitable genetic modification known in the art can be used to reduce endogenous cellobiose and take off The secretion activity of hydrogen enzyme.Such as, as the most described below, the modification imagined herein includes reducing by the fibre of described emiocytosis Tie up the modification of the amount of two glucocorticoid dehydrogenases.Also contemplate the modification of the amount reducing the cellobiose dehydrogenase expressed by described cell. Other embodiments include the modification reducing the transcriptional level of cellobiose dehydrogenase.Further embodiment includes compiling The disappearance wholly or in part of the gene of code cellobiose dehydrogenase.Other embodiments include reducing urging of cellobiose dehydrogenase Change the modification of effect.

Secretion enzyme. in some embodiments, the fungal cell of the present invention by genetic modification with reduce by described carefully The amount of the endogenous cellobiose dehydrogenase of intracrine.The minimizing of the amount of the cellobiose dehydrogenase of secretion can be to be secreted into born of the same parents The minimizing wholly or in part of the cellobiose dehydrogenase in external environment.The minimizing of the amount of the cellobiose dehydrogenase of secretion can be passed through Reduce the amount of the cellobiose dehydrogenase produced by described cell and/or by reducing the fibre that emiocytosis is produced by described cell The ability tieing up two glucocorticoid dehydrogenases realizes.Can be according to known in the art many for reducing the method for the ability of cell secretory polypeptide Plant any one of suitable method to carry out (see, e.g. Fass and Engels J.Biol.Chem., 271:15244- 15252 [1996], it is expressly incorporated herein by reference of text).Such as, the gene of Encoding Secreted Polypeptides can be modified with disappearance Secreting signal peptide or make it inactivate.In some embodiments, described fungal cell by genetic modification to destroy cellobiose The N end secreting signal peptide of dehydrogenase.In some embodiments, the amount by the cellobiose dehydrogenase of described emiocytosis is relative In the organism of the unmodified grown under essentially identical condition of culture or cultivate, the secretion of cellobiose dehydrogenase reduces At least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, About 98%, about 99% or more.

Additionally, in some embodiments, the total amount of cellobiose dehydrogenase activity is relative in essentially identical cultivation Under the conditions of grow or in the organism of unmodified cultivated, the total amount of the cellobiose dehydrogenase of secretion reduces at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, About 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% Or it is more.

The secretion of the cellobiose dehydrogenase reduced can be by known in the art for detecting protein or enzyme level Any one of multiple suitable method method determines.Such as, cellobiose dehydrogenase in the supernatant of fungal cultures Level can use Western blotting technique or utilize any other suitable protein of cellobiose dehydrogenase specific antibody Detection technique detects.Similarly, the secretion activity of the cellobiose dehydrogenase in the supernatant of fungal cultures can use such as this The mensuration for cellobiose dehydrogenase activity that literary composition is more fully described is measured.

Expression. in some embodiments, described fungal cell by genetic modification with reduce by described cell table The amount of the endogenous cellobiose dehydrogenase reached.As used herein, express refer to change into information coded in gene by The protein of this gene code.Therefore, reducing of the amount of the cellobiose dehydrogenase of expression represents the fibre finally translated by cell Tie up the minimizing of the amount of two glucocorticoid dehydrogenases.In some of such embodiment, the minimizing of expression is by reducing by encoding fiber disaccharide The amount of the mRNA of the genetic transcription of dehydrogenase realizes.In some other embodiments, the minimizing of expression is by reducing by encoding The amount of the protein of the mRNA translation of cellobiose dehydrogenase realizes.

The amount of the cellobiose dehydrogenase expressed by described cell is relative to cellobiose in the most adorned fungal cell The expression of dehydrogenase can be reduced at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, About 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more.In some of such embodiment, the minimizing of expression by In the organism of the unmodified grown under substantially the same condition of culture or cultivate, reduce by encoding fiber two glucocorticoid dehydrogenase The amount of the mRNA of genetic transcription realizes.

Additionally, in some embodiments, the minimizing of cellobiose dehydrogenase expression causes described fungal cell's Total expression of cellobiose dehydrogenase is relative to the unmodified grown under essentially identical condition of culture or cultivate Fungal cell reduces at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%.

The expression of the cellobiose dehydrogenase reduced can be by known in the art for detecting protein or enzyme level Any one of multiple method method determines.Such as, in the supernatant of fungal cultures, the level of cellobiose dehydrogenase can Use Western blotting technique or utilize any other suitable protein detection skill of cellobiose dehydrogenase specific antibody Art detects.

It is well known in the art for reducing the method for expression of polypeptides and multiple suitable side known in the art can be used Any one of method method is carried out.Such as, the gene of Encoding Secreted Polypeptides can be modified to destroy translation initiation sequence example Such as Shine-Delgarno sequence or Kozak consensus sequence.Additionally, the gene of Encoding Secreted Polypeptides can be modified with by frameshit Sudden change is incorporated in the transcripton of encoding endogenous cellobiose dehydrogenase.It will also be appreciated that be of little use, the use of codon can Cause the expression of polypeptides reduced.It will be appreciated that in some embodiments, the gene of encoding fiber two glucocorticoid dehydrogenase has at least one The individual nonsense mutation causing translating the protein of truncate.

The additive method of the amount of the polypeptide expressed by minimizing includes transcribing rear RNA silencing methods, such as antisense RNA and RNA Interference.Antisense technology is the nucleotide sequence fully set up, and include and use the nucleic acid array complementation with this gene.More specifically Ground, fungal cell to the expression of gene can by introduce with described nucleic acid array complementation can in described cell transcribed and energy It is enough that the nucleotide sequence of produced mRNA hybridization is reduced or eliminated with cell.Allowing complementary antisense base sequences Under conditions of hybridizing with described mRNA, therefore the amount of the albumen translated is reduced or eliminated.Method for antisence RNA is Known in the art (see for example, Ngiam et al., Appl Environ Microbiol., 66 (2): 775-82 [2000]；With Zrenner et al., Planta., 190 (2): 247-52 [1993]), be all expressly incorporated herein by reference of text this its).

Additionally, the modification of gene, lower or inactivate can through RNA disturb (RNAi) technology obtain (see for example, Kadotani et al. Mol.Plant Microbe Interact., 16:769 76 [2003], it is incorporated to by reference of text Herein).RNA interference method includes double-stranded RNA (dsRNA), short-hairpin RNA (shRNAs) and siRNA (siRNAs).Make Any suitable technology can be used to obtain by the potent silence of dsRNA (to see for example, Fire et al., Nature391:806-11 [1998]).Use shRNA silence be also fully established (see for example, Paddison et al., Genes Dev., 16:948-958[2002]).Use siRNA technology silence be also known (see for example, Miyagishi et al., Nat.Biotechnol.,20:497-500[2002]).The content of each above-mentioned list of references is incorporated to by reference of text Herein.

Transcriptional level. in some embodiments, the fungal cell of the present invention by genetic modification to reduce encoding endogenous The transcriptional level of the gene of property cellobiose dehydrogenase.As used herein, transcribe and similar term refers to be compiled in gene The information of code is converted into rna transcription.Therefore, the minimizing of the transcriptional level of cellobiose dehydrogenase is encoding fiber disaccharide dehydrogenation The minimizing of the amount of rna transcription of the RNA of enzyme.In some embodiments, transcriptional level is relative in essentially identical cultivation Under the conditions of grow or the transcriptional level of cellobiose dehydrogenase in the organism of unmodified cultivated reduces at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, About 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% Or it is more.

Additionally, in some embodiments, the minimizing of cellobiose dehydrogenase transcriptional level causes by described fungal cell Total cellobiose dehydrogenase of secretion is relative to the life by the unmodified grown under essentially identical condition of culture or cultivate Object reduce at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, About 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%.Transcribing of reducing can pass through the multiple side for detecting transcriptional level known in the art Any one of method determines.Such as, in fungal cell, the transcriptional level of specific mRNA can use quantitative RT-PCR technology or special Property detect specific mRNA other RNA detection techniques detect.Can be according to ability for subtracting the method for oligogenic transcriptional level Known to territory, any suitable method is carried out, and includes the partially or completely disappearance of gene, and to the destruction of gene promoter or Replace so that transcribing of this gene is reduced significantly or the most suppressed.Such as, the promoter of gene can be taken by weak promoter Generation (see for example, U.S. Patent No. 6,933, No. 133, it is expressly incorporated herein by reference of text).Therefore, if this weak startup Son is operatively connected with the coded sequence of endogenous polypeptide, then transcribing of this gene is reduced significantly or suppresses.

Gene delection. in some embodiments, the fungal cell of the present invention by genetic modification to lack at least in part Lose the gene of encoding endogenous cellobiose dehydrogenase.Generally, this disappearance reduce or eliminates and is secreted by described fungal cell The total amount of endogenous cellobiose dehydrogenase.In some embodiments, it is contemplated to the disappearance completely or the completeest of gene order Full disappearance.But, in order to reduce the amount of the endogenous cellobiose dehydrogenase secreted by fungal cell, deletion mutation need not be complete The full complete genome sequence removing encoding fiber two glucocorticoid dehydrogenase.Such as, in some embodiments, exist eliminate coding fibre Aminoacid, encoding secretion signals or coding in dimension disaccharide dehydrogenase activity site are at the endogenous secreted by described fungal cell The part of one or more nucleotide of the another part of the cellobiose dehydrogenase worked in cellobiose dehydrogenase activity Disappearance.

According to embodiment provided herein, the disappearance in the gene of encoding fiber two glucocorticoid dehydrogenase includes encoding described fibre Tie up the disappearance of one or more nucleotide in the gene of two glucocorticoid dehydrogenases.In some embodiments, there is encoding fiber two At least about the 5% of the gene of glucocorticoid dehydrogenase, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, About 95%, about 96%, about 97%, about 98%, about 99% or the disappearance of about 100%, wherein by the cellobiose dehydrogenase of emiocytosis Amount reduces.

Therefore, in some embodiments, disappearance causes the work of the cellobiose dehydrogenase secreted by described fungal cell Property relative to by the unmodified grown under essentially identical condition of culture or cultivate organism secrete cellobiose dehydrogenation The activity of enzyme reduces at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, About 96%, about 97%, about 98% or about 99%.

Additionally, in some embodiments, disappearance causes the total fiber two glucocorticoid dehydrogenase phase secreted by described fungal cell At least about 5% reduced for the most adorned fungal cell that grows under essentially identical condition of culture or cultivate, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, About 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%.

The disappearance of cellobiose dehydrogenase gene can be by the multiple method for detecting gene delection known in the art Any one detection and confirm, including the method in provided herein and embodiment.Such as, as exemplified here, gene lacks The genome area that mistake can use PCR amplification to modify confirms.It will be appreciated that the other suitable skill for confirming disappearance Art can use and be known, including southern blotting technique technology, the DNA sequencing of the genome area of modification and screening in restructuring The positive or negative mark mixed in event.

It is known for complete and/or excalation gene method, the fungal cell of genetic modification as herein described The multiple deletion method causing reducing the amount by the endogenous cellobiose dehydrogenase of this emiocytosis known in the art can be used Any one produce.This type of method can advantageously comprise use homology flanking markers standard gene destroy (see for example, Rothstein, Meth.Enzymol., 101:202-211 [1983], be expressly incorporated herein by reference of text).For gene delection Other technology include the PCR-based for shortage of standard method (see for example, Davidson et al., Microbiol., 148:2607 2615 [2002], is expressly incorporated herein by reference of text).

Other gene delection technology includes " male-female " box；Disappearance of based on cre/lox, increase homologous recombination Via Particle Bombardment Transformation and agriculture bacillus mediated gene disruption." male-female " method uses by a kind of mark for positive-selecting Thing gene and for feminine gender screening another marker gene composition box (see for example, Chang et al., Proc.Natl.Acad.Sci.USA84:4959–4963[1987]).Method based on Cre/lox uses and uses Cre recombinase Expression eliminate marker gene (see for example, Florea et al., Fung.Genet.Biol., 46:721-730 [2009]).

The method introducing DNA or RNA in fungal cell is well known by persons skilled in the art, includes but not limited to PEG Protoplast transformation, electroporation, via Particle Bombardment Transformation and the Agrobacterium-medialed transformation of mediation.Via Particle Bombardment Transformation uses a kind of work Skill, wherein DNA or RNA is introduced into cell on the granule of micron size, thus increases deletion construct passing to fungal cell Send (see for example, Davidson et al., Fung.Genet.Biol., 29:38-48 [2000]).Similarly, agriculture bacillus mediated Convert associating linear or isolate mark (split-marker) disappearance box and can help the deletion construct delivery (ginseng to target cell See such as, Wang et al., Curr.Genet., 56:297-307 [2010]).

Other method for gene delection wholly or in part includes, the destruction of gene.This type of gene disruption technique is Well known by persons skilled in the art, include but not limited to, insertion mutation, the integration (marked using and indicating of transposon Integration).However, it should be understood that provide any of the destruction of any other function aspects of coded sequence or gene Suitable technology can be used to produce the fungal cell of genetic modification provided herein.The method of insertion mutation can be according to this area Known any suitable method carries out (see for example, Combier et al., FEMS Microbiol.Lett., 220:141-8 [2003], it is expressly incorporated herein by reference of text).Additionally, agriculture bacillus mediated insertion mutation can be used to compile to insert to destroy The function of code gene such as destroys the sequence of any other function aspects of coded sequence or gene.

Transposon mutagenesis approach provides the another way for destroying gene.Transposon mutagenesis is to it is known in the art that And vivo techniques (see for example, Firon et al., Eukaryot.Cell2:247-55 [2003]) can be used；Or by using body Outer technology (see for example, Adachi et al., Curr Genet., 42:123-7 [2002]) is carried out；These lists of references each A piece is incorporated to by reference of text.Therefore, the gene disruption that can use the targeting of transposon mutagenesis destroys coding base to insert The sequence of the function of cause, such as destroys coded sequence or any other function aspects of gene.

The integration (REMI) of restriction enzyme-mediated is the another kind of method for gene disruption, and is well known in the art (see for example, Thon et al., Mol.Plant Microbe Interact., 13:1356-65 [2000], it is complete by quoting Literary composition is expressly incorporated herein).REMI produces insertion with random manner on surface in genomic restriction site, and some of them cause dashing forward Become.Therefore, inserting of the destruction in the gene showing encoding endogenous cellobiose dehydrogenase can be screened and utilize as provided herein Entering property mutant.

Catalytic destruction. in some other embodiments, described fungal cell by genetic modification to reduce cellobiose The catalytic effect of dehydrogenase.The minimizing of catalytic effect refers to as used standard technique provided herein or that this area is known per se to survey Amount, relative to the cellobiose dehydrogenase of unmodified, the minimizing of cellobiose dehydrogenase activity.Therefore, catalytic effect is reduced Genetic modification may result in such as, translated have enzymatic activity reduce protein.

The minimizing of catalytic effect is as used standard technique to measure, and cellobiose dehydrogenase activity is relative to unmodified Cellobiose dehydrogenase reduce about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, About 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more.In some other embodiments, as used standard technique to measure , genetic modification causes in terms of the total cellobiose dehydrogenase activity secreted by described fungal cell, cellobiose dehydrogenation Enzymatic activity reduces about 5% compared with the cellobiose dehydrogenase of unmodified, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, About 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%.

It is known for reducing the method for the catalytic effect of dehydrogenase, so, known in the art for reducing catalysis Any one of the multiple suitable method of effect can be used for genetic modification fungal cell provided herein.It is therefoie, for example, can heredity Modify fungal cell (to see, e.g. with the one or more residues in the avtive spot of inactivation cellobiose dehydrogenase Frederik et al.., Biochem., 42:4049 4056 [2003], be incorporated by herein by quoting).Such as, can repair Adorn one or more residue to reduce Binding Capacity, and/or one or more residue can be modified to reduce cellobiose dehydrogenase Catalysis activity.Therefore, can to electron acceptor (such as, the flavin) binding structural domain of cellobiose dehydrogenase or any its One or more residues in his substrate-binding domain carry out modifying to reduce or the catalysis effect of inactivation cellobiose dehydrogenase Power.Similarly, the allosteric of shape or activity that the residue mutations beyond avtive spot may result in cellobiose dehydrogenase changes, from And the catalytic effect reducing enzyme will be apparent from.

In some embodiments, other domains are targeted and suddenly change at least one, and it causes reducing endogenous fiber The catalytic effect of two glucocorticoid dehydrogenases.Such as, in some embodiments, the haemachrome binding structural domain to cellobiose dehydrogenase In the sudden change of one or more residues may result in minimizing catalytic effect (such as, Rotsaert et al., Arch.Biochem.Biophys., 390:206-14 [2001], it is expressly incorporated herein by reference of text).

Fungal cell

As indicated in this paper, the invention provides the fungal cell from cupreum section of section, described fungal cell by Genetic modification is to reduce by the amount of the endogenous cellobiose dehydrogenase activity of described emiocytosis, wherein said fungal cell's energy Enough secretions comprise the enzymatic mixture of cellulase.Cupreum section is Ascomycotina (Ascomycota), excrement shell Gammaproteobacteria (Sordariomycetes) section of the fungus in.Cupreum section of section include without hair Chaetomium, atresia Thielavia, hair beak shell belong to, Chaetomium, Corylomyces, rod softgel shell genus, Fa Shi shell genus, Thielavia, handle spore shell belong to and myceliophthora.Some embodiment party In case, the fungal cell of genetic modification provided herein is to belong to and the hair of Chaetomium selected from myceliophthora, Thielavia, rod softgel shell The member of shell Cordycepps.

In some embodiments, the fungal cell of genetic modification be selected from myceliophthora, Thielavia, rod softgel shell belong to and The phorozoon of the cupreum section member of Chaetomium or epigamous.In some embodiments, the fungal cell of genetic modification is selected from Sporotrichum, Chrysosporium, paecilomyces (Paecilomyces), Talaromyces and Acremonium.Also contemplate heredity to repair The fungal cell of decorations is further selected from belonging to Ctenomyces, thermophilic ascomycete belongs to and Scytalidium, fungal cells asexual belonged to including these Type and epigamous.In some embodiments, the fungal cell of genetic modification selected from addicted to cellulose side spore fusarium globosum shuttle mould, different, Different rod softgel shell bacterium, the bacterial strain of Tai Ruisi fusarium globosum shuttle, including its phorozoon and epigamous.Expection the invention is not restricted to cupreum section In any specific genus.In some other embodiments, the fungal cell of genetic modification is Acremonium, Arthroderma (Arthroderma), rod softgel shell genus, Thielavia, myceliophthora, thermophilic ascomycete genus, Chromocleista, the mould genus of silk clothes (Byssochlamys), Sporotrichum, Chaetomium, Chrysosporium, Scytalidium, Ctenomyces, paecilomyces or Talaromyces Thermophilic type species.It should be understood that for all aforementioned species, regardless of its kind name being referred to as, the fungus of the genetic modification shown herein Cell includes completely and the imperfect stage, and other taxonomy equivalents (such as, phorozoon) (see, e.g., Cannon, Mycopathol.,111:75-83[1990]；Moustafa et al., Persoonia14:173-175 [1990]；Upadhyay etc. People, Mycopathol., 87:71-80 [1984]；Guarro et al., Mycotaxon23:419-427 [1985]；Awao et al., Mycotaxon16:436-440[1983]；With von Klopotek, Arch.Microbiol., 98:365-369 [1974]).This Skilled person will readily appreciate that the identity of Suitable equivalents.Therefore, it should be understood that except as otherwise noted, otherwise originally In disclosure, the use of specific Nomination of species also refers to the species relevant by phorozoon or epigamous relation.Such as, following thing Kind be phorozoon or epigamous and therefore can be considered as synonym: thermophilic fungus destroyed wire, sporotrichum thermophile, sporotrichum thermophile, addicted to Cellulose side spore is mould, addicted to cellulose gold pityrosporion ovale, different rod softgel shell bacterium and different fusarium globosum shuttle.

In some embodiments, the fungal cell of genetic modification provided herein be produce cellulase fungus thin Born of the same parents.In some embodiments, the fungal cell producing cellulase expresses and the mixture of the eccrine fiber water-disintegrable enzyme of element.? In some embodiments, the fungal cell of genetic modification provided herein is the fungal cell from cupreum section of section, its secretion The water-disintegrable enzyme of two or more celluloses (such as, endoglucanase, cellobiohydrolase and/or β-glucosyl enzym). In some other embodiments, the fungal cell producing cellulase produces two kinds in these enzymes or many in any combination Kind.

It addition, in some embodiments, the fungal cell of genetic modification derives from the competent parent of lignocellulose Fungal cell.In some embodiments, the competent fungal cell of lignocellulose secretes one or more lignin peroxides Compound enzyme, manganese peroxidase, laccase and/or cellobiose dehydrogenase (CDH).

Present invention also offers the fungus training in the container being in the fungal cell comprising genetic modification described above Support thing.In some embodiments, this container comprises fluid medium, such as fermentation medium.Such as, this container can be to burn Bottle, biological treatment reactor or any suitable container.In some embodiments, this container comprises solid growth culture media. Such as, solid medium can be agar culture medium, such as potato dextrose agar, carboxymethyl cellulose, corn meal agar With any other suitable culture medium.In some embodiments, above-described fungal cell is the fungal cell separated.

Cellobiose dehydrogenase

As noted herein, term " cellobiose dehydrogenase " and " CDH " refer to cellobiose: acceptor 1-oxidoreduction Enzyme, this enzyme is catalyzed in the presence of acceptor and cellobiose is converted into cellobiose-1,5-lactone and the acceptor of reduction.Fine The example tieing up two glucocorticoid dehydrogenases falls in enzyme classification (E.C.1.1.99.18).Generally, such as ferrum, especially Fe (SCN) 3, molecule It is the same that oxygen, ubiquinone or cytochrome C may act as acceptor with other Polyphenols such as lignin, and 2,6-dichloroindophenols may act as connecing Receptor.The substrate of this enzyme include cellobiose, fiber-oligosaccharide, lactose and D-glucityl-1,4-β-D-MANNOSE, glucose, Maltose, mannobiose, sulfur are for cellobiose, galactosyl-mannose, 1,4-.beta.-Xylobiose, xylose.Electron donor is included in reduction end Hold β-Isosorbide-5-Nitrae-two hexose with glucose or mannose, but α-Isosorbide-5-Nitrae-hexoside, hexose, pentose and β-Isosorbide-5-Nitrae pentose are polymerized The substrate of at least some enzyme that thing may act as these enzymes (see, e.g. Henriksson et al., Biochim.Biophys.Acta Prot.Struct.Mol.Enzymol.,1383:48-54[1998]；With Schou et al., Biochem.J., 330:565-571 [1998])。

In some embodiments, CDH enzyme comprises the N knot of conservative glucose-methanol-choline (GMC) oxidoreductase Both C-structure territories of structure territory and GMC oxidoreductase.In some other embodiments, CDH comprises only GMC oxidoreductase N domain.GMC oxidoreductase be FAD flavoprotein oxidoreductase (see for example, Cavener, J.Mol.Biol., 223:811-814[1992]；And Vrielink and Blow, Biochem., 32:11507-15 [1993]).GMC oxidoreduction Enzyme includes multiple proteins, such as choline dehydrogenase, methanol oxidase and cellobiose dehydrogenase (CDH), and it has has sequence Several regions of row similarity.As defined according to entry GMC_oxred_N (PF00732) by Pfam database root, these districts Be positioned in N end portion region in territory corresponds to FAD ADP-binding structural domain.Similarly, according in Pfam data base C end conserved domain (GMC oxidoreductase C-structure is defined according to what entry GMC_oxred_C (PF05199) was listed Territory).

Cellobiose dehydrogenase can be divided into Liang Ge family.First family comprises catalysed partial and the second family comprises catalytic portions Divide and cellulose binding motif (CBM).The 3-dimensional structure of exemplary fiber two glucocorticoid dehydrogenase is characterized with two globular domains, Each comprises in two cofactors, i.e. haemachrome or flavin.Avtive spot gap between two domains In.The oxidation of cellobiose generally goes through the 2-electron transfer from cellobiose to flavin and occurs, and produces cellobiose-1, in 5- Ester and the flavin of reduction.Activity FAD is regenerated by the electron transfer to heme group, leaves the haemachrome being reduced.My god So the haemachrome of state passes through the reaction regeneration at the second avtive spot with oxidized form substrate.In some embodiments, connect Receptor is preferably the phenolic compounds such as dichloroindophenol (DCIP) of cyanogen ferrum ferrum, cytochrome C or oxidation, and one is generally used for ratio The acceptor that color measures.Metal ion and O2 are also acceptors, but for major part cellobiose dehydrogenase, cellobiose oxygen Change enzyme is compared to ferrum or the low several quantity of the viewed response speed of organic oxidizing agent for the response speed of these acceptors Level.After the release of cellobiose lactone, product can experience spontaneous open loop and (see, e.g. Hallberg etc. to produce cellobionic acid People, J.Biol.Chem., 278:7160-7166 [2003]).It will be understood by those skilled in the art that cellobiose dehydrogenase activity Generally utilize oxygen or the existence of oxidoreduction acceptor of equal value, described oxidoreduction acceptor of equal value can be such as lignin, Molecular oxygen, cytochrome C, redox dye, benzoquinone and/or Fe²⁺Complex.

Cellobiose dehydrogenase activity can use any one of multiple suitable method known in the art method to survey Amount (see, e.g. Combier et al., Biochem J., 220:565-71 [1998], it is incorporated to by reference of text).Example As, can be active to DCPIP (2,6-chlorophenesic acid indole by the absorbance monitoring CDH in the presence of cellobiose under 530nm Phenol) reduction.

As provided herein, usual with the secreting active fungal cell reducing cellobiose dehydrogenase by genetic modification There is the secretion activity of the endogenous cellobiose dehydrogenase of minimizing.Therefore, from fungal species each of described herein One or more cellobiose dehydrogenases can be by genetic modification institute targeting.In some embodiments, cellobiose dehydrogenase comes Fungal species from cupreum section of section.

Table 1 below lists from cupreum section member authentication to some examples of cellobiose dehydrogenase.Real at some Executing in scheme, cellobiose dehydrogenase is from selected from following fungal species: addicted to cellulose side spore fusarium globosum shuttle mould, different, different capsule Shell bacterium, Tai Ruisi fusarium globosum shuttle, chaetomium globosum and thermophilic fungus destroyed wire.Following table lists from these species identification to some are fine Tie up two glucocorticoid dehydrogenases.The protein listed in following table is known in the art or is accredited as the fiber of cellobiose dehydrogenase herein The example of two glucocorticoid dehydrogenases.

* the registration number for Tai Ruisi fusarium globosum shuttle represents USDOE (DOE) Polymorphism group academy (JGI) Genome sequence.

There is provided herein some aminoacid sequences of encoding fiber two glucocorticoid dehydrogenase.Such as, coding thermophilic fungus destroyed wire CDH1 Nucleotides sequence be listed in and list with SEQ ID NO:1 herein, and the aminoacid sequence of the coding of thermophilic fungus destroyed wire CDH1 is with SEQ ID NO:2 lists.

In some embodiments, cellobiose dehydrogenase is cellobiose dehydrogenase EC1.1.99.18.Implement at some In scheme, cellobiose dehydrogenase is the fibre of the aminoacid sequence with thermophilic fungus destroyed wire CDH1 listed such as SEQ ID NO:2 Tie up two glucocorticoid dehydrogenases.In some other embodiments, cellobiose dehydrogenase comprises any one registration number listed in table 1 GenBank entry in provide aminoacid sequence.In some embodiments, cellobiose dehydrogenase by with SEQ ID NO: 1 at least about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, About 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, Coded by about 97%, about 98%, about 99% or about 100% identical nucleotide sequence.In some embodiments, cellobiose dehydrogenase By the aminoacid sequence listed such as SEQ ID NO:2 with coding or the Genbank bar of any one registration number listed at table 1 In mesh provide the nucleotide sequence at least about 60% of aminoacid sequence, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, About 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, Coded by about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical nucleotide sequence. In some embodiments, cellobiose dehydrogenase is by can be with SEQ ID NO:1 described above medium strict or strict Under the conditions of the nucleic acid sequence encoding that optionally hybridizes.In some embodiments, cellobiose dehydrogenase by can with coding SEQ In the Genbank entry of any one registration number that ID NO:2 or table 1 are listed provide aminoacid sequence nucleotide sequence in Deng the nucleic acid sequence encoding strictly or optionally hybridized under stringent condition.

In some embodiments, cellobiose dehydrogenase comprise with such as the aminoacid sequence listed in SEQ ID NO:2, Or the aminoacid sequence provided in the Genbank entry of any one registration number listed at table 1 has at least about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, About 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, About 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or the phase of about 100% Aminoacid sequence like property.Cellobiose dehydrogenase sequence can be by any one of multiple method known in the art method Determine.Such as, can be for data base, such as ncbi database carries out sequence alignment, and can be chosen with minimum HMM E-value Sequence.

In some embodiments, the fungal cell of the present invention by genetic modification with reduce by the two of described emiocytosis The amount of the cellobiose dehydrogenase activity of kind or more kinds of endogenous cellobiose dehydrogenase.In some embodiments, described The first in two or more cellobiose dehydrogenases comprises and SEQ ID NO:2 at least about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, About 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% are identical The second in aminoacid sequence, and the two or more kinds of cellobiose dehydrogenase comprises with SEQ ID NO:2 at least about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, About 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, About 98% or about 99% identical aminoacid sequence.

Enzymatic mixture

The enzyme mixing that comprise at least one that by fungal cell express or multiple cellulose water-disintegrable enzyme is also provided herein Thing, as described herein, described fungal cell by genetic modification to reduce by the endogenous cellobiose of described emiocytosis The amount of dehydrogenase activity.Cellulase is produced by various microorganisms.Cellulose from filamentous fungi He some antibacterials Enzyme (and hemicellulase) is widely utilized in the many commercial Application relating to that natural fiber is processed saccharogenesis.Contemplate The mixture of any enzyme listed herein will can use in the present invention.

In some embodiments, described enzymatic mixture comprises at least one expressed by fungal cell or multiple cellulose Water-disintegrable enzyme, as described herein, described fungal cell is fine to reduce by the endogenous of described emiocytosis by genetic modification The amount of dimension disaccharide dehydrogenase activity.In some embodiments, described fungal cell is wooden from utilizing of cupreum section of section The cell of cellulose.In some embodiments, the fungal cell of genetic modification provided herein is selected from myceliophthora, shuttle spore Shell belongs to, rod softgel shell belongs to or the member of the cupreum section of Chaetomium.In some other embodiments, the fungal cell of genetic modification Can also is that and belong to selected from myceliophthora, Thielavia, rod softgel shell or the phorozoon of cupreum section member of Chaetomium or epigamous. It addition, the fungal cell of genetic modification is further selected from Sporotrichum or Acremonium or Talaromyces.Also contemplate genetic modification Fungal cell selected from Ctenomyces, thermophilic ascomycete belong to and Scytalidium, including the fungal cell belonged to from these phorozoon and Epigamous.In some embodiments, fungal cell is selected from addicted to cellulose side spore fusarium globosum shuttle mould, different, different rod softgel shell bacterium, too This fusarium globosum shuttle auspicious, chaetomium globosum, the basket bacterium of handle and the species of thermophilic fungus destroyed wire, including its phorozoon and epigamous.

In addition to above-mentioned enzyme, other enzymes such as laccase can be used in the mixture of the present invention.Laccase is in many In plant, fungus and microorganism find containing CuO-2 layer.Act on phenol and similar molecule laccase enzymatic and carry out an electricity Son oxidation.Laccase can be polymeric form and enzyme form of living can be dimer or trimer.

Mn dependency peroxidase can be also used in the mixture of the present invention.Mn's dependency peroxidase (MnP) Enzymatic activity depends on Mn²+.It is not bound by theory ground, it has been suggested that the Main Function of this enzyme is by Mn²⁺It is oxidized to Mn³⁺(see example As, Glenn et al. Arch.Biochem.Biophys., 251:688-696 [1986]).Subsequently, phenol substrate is by produced Mn³⁺ Oxidation.

Lignin peroxidase can be also used in the mixture of the present invention.Lignin peroxidase is that catalysed in vitro gathers The outer haemachrome of born of the same parents of the oxidisability depolymerization closing the weak solution of state lignin.Some substrates of LiP, the most famously 3,4-dimethoxy Benzylalcohol (veratryl alcohol, VA) is the activated redox compound being asserted and having served as redox mediator.VA is at yellow spore The lignin of the flat lead fungi of raw wool cracks the secondary metabolite produced while culture produces LiP and is not bound by theory ground, Have been pointed out its in vivo LiP-catalysis lignin oxidation in serve as physiological oxidation reducing medium (see for example, Harvey et al., FEBS Lett.195:242–246[1986])。

In some embodiments, it can be favourable for utilizing acellular enzymatic mixture.Acellular enzymatic mixture is usual Comprise from any cell, including the enzyme of the cell separation of secretase.Acellular enzymatic mixture can use known in the art Prepared by any one of multiple suitable method (such as filtering or centrifugal) method.In some embodiments, enzymatic mixture It is the most acellular, the most acellular or the most acellular.

In some embodiments, present in cellulase mixture two or more cellulase and any additionally Enzyme by the fungal cell of single genetic modification or by combination or the individually different microorganisms secretion in fermentation.Similarly, Present in cellulase mixture, two or more cellulase and any other enzyme can be by the differences of different organisms Bacterial strain express individually or one-tenth group express, and these enzymes by external merging to prepare cellulase mixture.It is contemplated within Cellulase in enzymatic mixture is expressed with any other enzyme individually by the different strains of single creature body or is become group Ground is expressed, and these enzymes are combined to prepare cellulase mixture.

In some embodiments, described enzymatic mixture comprises at least one expressed by fungal cell or multiple cellulose Water-disintegrable enzyme, as described herein, described fungal cell by genetic modification to reduce by the endogenous fiber of described emiocytosis The amount of disaccharide dehydrogenase activity.In some embodiments, described fungal cell is to utilize wooden fibre from cupreum section of section The cell of dimension element.In some embodiments, the fungal cell of genetic modification provided herein is selected from myceliophthora, shuttle spore shell The member of the cupreum section of genus, rod softgel shell genus and Chaetomium.The fungal cell of genetic modification can also is that selected from myceliophthora, shuttle The phorozoon of the cupreum section member of spore shell genus, rod softgel shell genus and Chaetomium or epigamous.It addition, the fungal cell of genetic modification It is further selected from Sporotrichum, Acremonium, Ctenomyces, Scytalidium and thermophilic ascomycete to belong to, thin including the fungus belonged to from these The phorozoon of born of the same parents and epigamous.In some embodiments, fungal cell be selected from addicted to cellulose side spore fusarium globosum shuttle mould, different, Different rod softgel shell bacterium, Tai Ruisi fusarium globosum shuttle, chaetomium globosum, the basket bacterium of handle and the species of thermophilic fungus destroyed wire, including its phorozoon and Epigamous.

In some embodiments, the cellulase mixture of the present invention produces in fermentation technology, the most described herein Fungal cell be cultured in deep-layer liquid cultivation and fermentation thing.In some embodiments, use in batches, fed-batch or company The deep fermentation thing of fungal cell is hatched in continuous process.In batch process, in addition to the oxygen for aerobic technique, all Required material is placed in the reactor and allows fermentation to carry out when operation starts until terminating, and checks and accepts in the time terminated Collection product.In some embodiments, for producing the batch process of the enzymatic mixture of the present invention at shaking flask or bioreactor In carry out.In some embodiments using fed-batch processes, to culture, continuously or sequentially supply is a kind of or many Plant nutrient media components and do not remove culture fluid.In a continuous process, supply fresh culture with speed equal on volume and connect Continuous culture fluid of removing is to be maintained at culture under the stable speed of growth.It will be understood by those skilled in the art that fermentation medium Normally liquid, and comprise carbon source, the nitrogen source that can be added into fermentation medium and produce with the growth and enzyme improving fungal cell And other nutritional labelings, vitamin and mineral.These other nutrient media components can use fungal cell's inoculum culture Before, concurrently or afterwards add.

In some embodiments of the technique of the enzymatic mixture for producing the present invention, carbon source comprises inducing fungal thin The carbohydrate of cellular expression cellulase.Such as, in some embodiments, described carbon source comprise cellulose, cellobiose, Sophorose, xylan, xylose, 1,4-.beta.-Xylobiose and/or known cellulase induction and β-glucosyl enzym are expressed in these fungal cells Relevant oligosaccharide or one or more of polysaccharide.In some embodiments utilizing batch fermentation, carbon source is in inoculation Before or concurrently join fermentation medium.In some embodiments utilizing fed-batch fermentation or continuous operation, Carbon source is continually or intermittently supplied during fermentation technology.Such as, in some embodiments, with about 0.2g carbon/L culture/h The carbon feed rate supply carbon source of any suitable amount between about 2.5g carbon/L culture/h or therebetween.

Can carry out at any suitable temperature for producing the method for the enzymatic mixture of the present invention, generally from about 20 DEG C to About 100 DEG C or the most any suitable temperature, such as from about 20 DEG C to about 80 DEG C, 25 DEG C to about 65 DEG C or the most any properly Temperature, or from about 20 DEG C, about 22 DEG C, about 25 DEG C, about 26 DEG C, about 27 DEG C, about 28 DEG C, about 29 DEG C, about 30 DEG C, about 32 DEG C, about 35 DEG C, about 37 DEG C, about 40 DEG C, about 45 DEG C, about 50 DEG C, about 55 DEG C, about 60 DEG C, about 65 DEG C, about 70 DEG C, about 75 DEG C, about 80 DEG C, about 85 DEG C, about 90 DEG C, about 95 DEG C and/or the most any suitable temperature.

Can carry out under any suitable pH for producing the method for the enzymatic mixture of the present invention, generally from about 3.0 to 8.0 or the most any suitable pH, such as from the suitable pH of about pH3.5 to pH6.8 or the most any, such as from about pH3.0, About 3.2, about 3.4, about 3.5, about 3.7, about 3.8, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.2, about 5.4, about 5.5, about 5.7, about 5.8, about 6.0, about 6.2, about 6.5, about 6.8, About 7.0, about 7.2, about 7.5, about 8.0 or the most any suitable pH.

In some embodiments, enzymatic mixture is comprised in the fungal cell's containing genetic modification as described herein In container.In some embodiments, this container comprises fluid medium.In some embodiments, this container is flask, life Thing treatment reactor or any other suitable container.In some embodiments, described enzymatic mixture is with liquid meter.? In some embodiments, liquid volume can be greater than about 0.01mL, about 0.1mL, about 1mL, about 10mL, about 100mL, about 1000mL or Greater than about 10L, about 50L, about 100L, about 200L, about 300L, about 400L, about 500L, about 600L, about 700L, about 800L, about 900L, about 1000L, about 10,000L, about 50,000L, about 100,000L, about 250,000L, about 500,000L or greater than about 1, 000,000L。

In some embodiments, after fermentation, use and comprise the fermentation medium of fungal cell, or use that to comprise fungus thin Born of the same parents and the fermentation medium of enzymatic mixture, or such as pass through to filter or be centrifuged to make enzymatic mixture separate with fungal cell, and make With the enzymatic mixture in fermentation medium.In some embodiments, low molecule solute such as fermentation medium is not consumed Component is removed by ultrafiltration.In some embodiments, enzymatic mixture by evaporating, precipitate, settle, filter or any suitably Means concentrate.In some embodiments, chemical substance such as glycerol, sucrose, sorbitol etc. are added with stable enzymatic mixture.? In some embodiments, in enzymatic mixture, add other chemicals such as sodium benzoate or potassium sorbate to prevent microorganism dirty The growth of dye thing.

For the method producing glucose

Present invention also offers the technique for producing glucose, connect with enzymatic mixture described herein including making cellulose Touch.Such as, in some embodiments, this technique comprises and makes cellulose and comprise the water-disintegrable enzyme of two or more celluloses Enzymatic mixture contacts, and at least one of the wherein said water-disintegrable enzyme of two or more celluloses is thin by fungus as described herein Cellular expression.In some embodiments, the method using enzymatic mixture to produce glucose from cellulose is batch hydrolysis, continuous water Solve or a combination thereof.In some embodiments, hydrolysis is that be stirred, unmixing or a combination thereof.

For produce from cellulose glucose method can in any suitable temperature, including about 30 DEG C and about 80 DEG C it Between, or the most any suitable temperature, e.g., from about 30 DEG C, about 35 DEG C, about 40 DEG C, about 45 DEG C, about 50 DEG C, about 55 DEG C, about 60 DEG C, About 65 DEG C, about 70 DEG C, about 75 DEG C, the temperature of about 80 DEG C or the most any suitable temperature and the pH of about 3.0 to about 8.0, or its Between any suitable pH, such as in about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, carry out under the pH of about 8.0, or the most any suitable pH.Hydrolysis start before cellulose the denseest in hydrolysis reactor Degree is preferably about 0.1% (w/w) to about 15% (w/w) or any suitable amount therebetween, such as, about 2%, about 4%, about 6%, about 8%, about 10%, about 12%, about 14%, about 15% or any suitable amount therebetween.

The dosage of cellulase mixture can be every gram of cellulose about 0.1 to about 100mg protein, or the most any conjunction Suitable amount, such as every gram cellulose about 0.1, about 0.5, about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, About 60, about 70, about 80, about 90, about 100mg protein or the most any suitable amount.Hydrolysis can carry out about 0.5 hour to about The time period of 200 hours, or the most any suitable time.Such as, in some embodiments, hydrolysis carries out about 15 hours extremely The time period of about 100 hours, or the most any suitable time, or its can carry out about 0.5 hour, about 1 hour, about 2 hours, about 4 hours, about 8 hours, about 12 hours, about 15 hours, about 20 hours, about 25 hours, about 30 hours, about 35 hours, about 40 hours, About 45 hours, about 50 hours, about 55 hours, about 60 hours, about 65 hours, about 70 hours, about 75 hours, about 80 hours, about 85 Hour, about 90 hours, about 95 hours, about 100 hours, about 120 hours, about 140 hours, about 160 hours, about 180 hours, about 200 hours or the most any suitable time.Should be understood that reaction condition is not meant to limit the present invention in any manner and can be by Those skilled in the art adjust as desired.

In some embodiments, enzymatic hydrolysis is generally carried out in hydrolysis reactor.Enzymatic mixture is by pretreatment Lignocellulosic material (also referred to as " substrate ") is added to substrate before, during or after adding to hydrolysis reactor.

In the method making cellulosic material contact with enzymatic mixture, can arbitrary according to multiple method known in the art Plant and adjust multiple environmental condition the formation of hydrolyzate such as glucose to be maximized.Such as, temperature, pH, % dissolved oxygen and stir Mix speed each can be individually adjusted.In some embodiments, enzymatic mixture is cell-free mixture as described herein.

As described herein, the enzymatic mixture of the cellobiose dehydrogenase activity with minimizing is used to produce the side of glucose Method causes the corresponding technique than using the enzymatic mixture with the cellobiose dehydrogenase activity fully supplemented higher from enzyme Promote the glucose yield of the cellulose of hydrolysis.It addition, these methods cause the cellobiose product of the minimizing in enzymic hydrolysates It is converted to oxidation product.

In some embodiments of the method for the cell and/or enzymatic mixture that use genetic modification provided herein, can Measure the glucose yield improved the most quantitatively.As described herein, glucose yield can be according to every theoretical maximum glucose yield institute Produce glucose amount or according to Gmax describe.It will be understood by those skilled in the art that as computational theory value such as Gmax and reason During the maximum glucose yield of opinion, two hydrogen atoms and an oxygen added in hydrolysis reaction on glucose molecule are counted The quality of atom.Such as, when the polymer of " n " individual glucose unit is hydrolyzed, the water of (n-1) unit is added in hydrolysis On the glucose molecule formed, so the weight many about 10% of the cellulose consumed in the weight ratio hydrolysis of produced glucose (such as, hydrolysis 1g cellulose will produce about 1.1g glucose).Accordingly, as citing, if there is 5g when hydrolysis starts Total usable fibers element, and after reaction, remain 2g residual cellulose, the most total cellulose being hydrolyzed is 3g cellulose.This is anti- Under the conditions of Ying, the theoretical maximum glucose yield of 100% (w/w) be about 5.5g glucose.Gmax based on reaction in by hydrolysis quilt Release or the 3g cellulose converted calculate.Therefore, in this example, the Gmax of 100% (w/w) is about 3.3g glucose.Cellulose Level, total available quantity or unhydrolysed or residual cellulose amount can be by multiple conjunctions known in the art present in substrate Any one of suitable method quantitatively, such as, passes through IR spectroscopy or by measuring the glucose that Concentrated acid hydrolysis cellulose produces Amount (see, e.g. U.S. Patent No. 6,090,595 and 7,419, No. 809, it is all expressly incorporated herein by reference of text).

Such as, in some embodiments, content of cellulose passes through acid hydrolyzed cellulose, followed by concentration of glucose Determine, count water necessary to hydrocellulose and determine and (see for example, U.S. Patent No. 6,090,595 and 7,419,809 Number).In an example, the slurry of centrifugal raw material, wash with water, and be suspended in the sulphuric acid that clean sulfuric acid concentration is 70%.? Hatch slurry at 40 DEG C 30 minutes, be diluted to 2% sulphuric acid the most in deionized water.At this time point, to sample at 121 DEG C Product steam high-voltage sterilizing 1 hour, to change into monomeric glucose by oligomer.Concentration of glucose is by HPLC or any suitably Enzymatic determination is measured.In some alternate embodiment, content of cellulose is by infrared spectrum credit as described in Example 1 Analysis.Such as, washable solid and be placed on Infrared Spectroscopy detection crystal on and measure between 500-4000cm-1 Absorbance.

Glucose level quantitatively (can see example by any one of multiple suitable method known in the art method As, U.S. Patent No. 6,090,595 and 7,419, No. 809).Such as, can use based on glucoseoxidase and Radix Cochleariae officinalis peroxidating The enzyme coupling of thing enzyme measures and determines that concentration of glucose (see for example, Trinder, Ann.Clin.Biochem., 6:24 27 [1969], it is expressly incorporated herein by reference of text).The method of other glucose quantitation includes that chromatography (see for example, U.S. State's patent the 6,090,595th and 7,419,809).Cellobiose level can be by multiple conjunction well known by persons skilled in the art Any one of suitable HPLC method measure (see, e.g. Kotiranta et al., Appl.Biochem.Biotechnol., 81: 81-90 [1999]), it is expressly incorporated herein by reference of text).

Similarly, cellobiose and glucose products turning to oxidation product such as cellobiose lactone and gluconolactone The minimizing changed can be quantitative by any one of multiple suitable method known in the art method.Such as, glucose or fibre Tie up two glycoxidative products to use infrared spectroscopy or by chromatographic process such as HPLC detection and quantitatively (see, e.g. Rakotomanga et al., J.Chromatog.B., 4:277-284 [1991]；With Mansfield et al., Appl.Environ.Microbiol., 64:3804-3809 [1997], it is all expressly incorporated herein by reference of text).Therefore, The letter of total oxidation product of such as every theoretical maximum glucose yield by glucose or total oxidation of cellobiose, can be defined as Number or the function of Gmax.

Cellulosic material

Any material comprising cellulose can be used for the present invention.Main polysaccharide in the primary cell wall of biomass is fiber Element, second abundant be hemicellulose, the 3rd is pectin.The secondary cell wall produced after cell has terminated growth also comprises Polysaccharide is also strengthened by the polymeric lignin with hemicellulose covalent cross-linking.Cellulose is the homopolymer of anhydro cellobiose, therefore It is linear β-(1-4)-D-glucosan, and hemicellulose includes being in complex branches structure multiple with a series of substituent group Compound, such as xylan, xyloglucan, arabinoxylan and mannan.Although typically multiform, cellulose exists In plant tissue, the insoluble crystal substrate mainly as parallel dextran chain occurs.Hemicellulose generally with hydrogen bonding in Cellulose and other hemicelluloses, cell wall matrix is stablized in this help.

Cellulose is commonly found in, such as, the stem of plant, leaf, shell (hull), shell (husk) and cob (cob) or tree In leaf, branch and timber.Cellulosic material can be but not limited to, and herbaceous material, agricultural residue, forestry residue, municipal administration are solid Body waste material, waste paper and paper pulp and paper mill residue (see, e.g. Wiselogel et al., at Charles E.Wyman, (compile Volume),Handbook on BioethanoL, Taylor&Francis, Washington D.C. [1995], at the 105-118 page In；Wyman,Biores.Technol.,50:3-16[1994]；Lynd,Appl.Biochem.Biotechnol.,24/25: 695-719[1990]；And Mosier et al., Adv.Biochem.Eng.Biotechnol., 65:23-40 [1999]).Ying Li Solve, in some embodiments, cellulose is wood fibre prime form, lignocellulose be mixing substrate in comprise wooden Element, cellulose and/or the Plant cell wall material of hemicellulose.In some embodiments, cellulosic material is wood fibre Element.

The lignocellulosic material of pretreatment is the material of plant origin, comprises the fiber of at least 10% before pre-processing Element (dry weight), the cellulose of more preferably more than about 30%, the cellulose of even more preferably greater than 40%, e.g., from about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, About 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, About 49%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or the most any Suitably percentage ratio, and at least about 10% (dry weight) or the lignin of at least about 12% (dry weight), and carried out physics and/or change Process so that fiber be more easy to by cellulase close to and/or be more easily accepted by the effect of cellulase.In some embodiments In, lignocellulosic material can comprise higher levels of cellulose after the pre-treatment.Such as, if using low-kappa number, then partly Cellulosic component is hydrolyzed, and which increases the relative level of cellulose.In this case, the raw material of pretreatment can comprise and is more than The cellulose of about 20% and the lignin of greater than about 12%.

The lignocellulosic material that can be used for the present invention includes but not limited to, agricultural residue, such as corn straw, wheat Stalk, barley-straw, Caulis et Folium Oryzae, Herba bromi japonici stalk, Rape Straw, sugarcane stem and soybean stalk；Fibre technology residue, such as corn fiber, sweet Puree, pulp mill's fines and waste residue or bagasse；Forestry residue, such as poplar, other hardwoods, soft wood and sawdust； Or grass such as switchgrass, Miscanthus, Value of Spartina Anglica and reed canary grass.In some embodiments, lignocellulosic material first passes through and includes But be not limited to any one of following multiple method stand size and reduce: mill, grind, vibrate, tear up, suppress/expand and/ Or other kinds of mechanism.Reduce to enter by adapting to any kind of equipment of this purpose by the size of mechanism OK, such as but not limited to, beater grinder.

Pretreatment

In some embodiments, the substrate of enzymatic mixture includes the cellulosic material of pretreatment.It is therefoie, for example, at this In the certain methods that literary composition describes, any pretreating process known in the art can be used for destroying the plant cell wall of cellulosic material Component (see for example, Chandra et al., Adv.Biochem.Engin.Biotechnol., 108:67-93 [2007]；Galbe And Zacchi, Adv.Biochem.Engin.Biotechnol., 108:41-65 [2007]；Hendriks and Zeeman, Biores.Technol.,100:10-18[2009]；Mosier et al., Biores.Technol., 96:673-686 [2005]； Taherzadeh and Karimi, Int.J.Mol.Sci., 9:1621-1651 [2008]；And Yang and Wyman, Biofuels Bioprod.Bioref.Biofpr.2:26-40[2008]；It is all expressly incorporated herein by reference of text).

In some embodiments, before pre-processing, any one using multiple proper method known in the art is right Cellulosic material carries out particle size minimizing, pre-soaking, humidifying, washing or conditioning.Conventional pretreatment includes but not limited to, steams Vapour pretreatment (with or without explosion), dilute acid pretreatment, hot-water pretreatment, oxygenation pretreatment, Calx preconditioning, wet oxidation, Wet explosion, ammonia filament expansion, dilute ammonia pretreatment, organic solvent pretreatment and Biological Pretreatment.Other pretreatment includes that ammonia oozes Thoroughly, ultrasonic, electroporation, microwave, supercritical CO₂, supercritical H₂O, ozone and gamma-radiation pretreatment.In some embodiments, fine Dimension cellulosic material is pretreated before hydrolysis and/or fermentation.In some embodiments, pretreatment is entered the most before hydrolysis OK.In some alternate embodiment, pretreatment and enzyme hydrolysis carry out discharging fermentable saccharide simultaneously, such as glucose, Xylose and/or cellobiose.In some embodiments, pre-treatment step itself causes biomass to fermentable saccharide A little conversions, even if there be no enzyme.

Steam pre-treatment.In steam pre-treatment, heating cellulose material is to destroy the component of plant cell wall, including wood Quality, hemicellulose and cellulose are so that cellulose and other fraction (such as, hemicellulose) are more easy to by enzyme close.Cellulose Material is passed to reaction vessel or through reaction vessel, injects steam so that temperature to be increased to required temperature in reaction vessel Degree and pressure and keep steam in reaction vessel, continue the desired response time.In some embodiments, steam pre-treatment Preferably carry out at about 140 DEG C to about 230 DEG C, and in other embodiments, it carried out at about 160 DEG C to about 200 DEG C, And in further embodiment, it is carried out at about 170 DEG C to about 190 DEG C, wherein optimum temperature range depends on that chemistry is urged Any interpolation of agent.In some embodiments, the time of staying of steam pre-treatment is about 1 to about 15 minute, and at other In embodiment, it is about 3 to about 12 minutes, and the most in other embodiments, and it is about 4 to about 10 minutes, the most most preferably The time of staying depend on any interpolation of temperature range and chemical catalyst.Steam pre-treatment allows relatively high solid to bear Carry, so that cellulosic material mostly just moistens in preprocessing process.Steam pre-treatment often with pretreatment after to material Explosion release combination, it is referred to as steam explosion, it is, rapid flash to atmospheric pressure and material turbulent flow with increase fermentation can The surface area touched (see for example U.S. Patent No. 4,451,648；Duff and Murray, Biores.Biophys., 855:1-33[1996]；Galbe and Zacchi, Appl.Microbiol.Biotechnol., 59:618-628 [2002]；And U.S. Patent Application Publication the 2002/0164730th, it is all expressly incorporated herein by reference of text).During steam pre-treatment, Cleaved and gained the sour autocatalysis hemicellulose fraction of the acetyl group of hemicellulose is hydrolyzed into monosaccharide and oligosaccharide.Only exist Lignin is removed on limited extent.Catalyst such as H2SO4 or SO2 (w/w of typically about 0.3 to about 3%) is generally pre-at steam Add before processing, which reduce time and temperature, add recovery, and improve enzymatic hydrolysis (see for example, Ballesteros et al., Appl.Biochem.Biotechnol., 129/-132:496-508 [2006]；Varga et al., Appl.Biochem.Biotechnol.,113-116:509-523[2004]；And Sassner et al., Enz.Microb.Technol.,39:756-762[2006])

Chemical Pretreatment: term " chemical treatment " refer to promote cellulose, hemicellulose and/or the separation of lignin and/ Or any Chemical Pretreatment of release.The suitably example of Chemical Pretreatment technique includes, but not limited to dilute acid pretreatment, dilute Oxygenation pretreatment (see for example, U.S. Patent Application Publication the 2007/0031918th and 2007/0037259), Calx preconditioning, Wet oxidation, ammonia fiber/freezing explosion or expansion (AFEX), ammonia are impregnated with (APR), dilute ammonia pretreatment and organic solvent pretreatment (see for example, WO2006/110891, WO2006/11899, WO2006/11900 and WO2006/110901).

In dilute acid pretreatment, by cellulosic material and diluted acid, it is common that H2SO4 and water mix to form slurry, steam It is heated to preferred temperature, after the time of staying, quickly becomes atmospheric pressure.Dilute acid pretreatment can carry out (example with many reactor designs Such as, plug flow reactor, flow reactor or Continuous convective are shunk bed reactor) (see for example, Duff and Murray, on Literary composition；Schell et al., Biores.Technol., 91:179-188 [2004]；And Lee et al., Adv.Biochem.Eng.Biotechnol.,65:93-115[1999])。

In some embodiments, with calcium carbonate, sodium hydroxide or ammonia at the low temperature and about 1 of about 85 DEG C to about 150 DEG C Hour carry out Calx preconditioning (Wyman et al., Biores.Technol., 96:1959-1966 to time of staying of a couple of days [2005]；With Mosier et al., Biores.Technol.96:673-686 [2005]).

Wet oxidation is under the oxygen adding oxidant such as hydrogen peroxide or superpressure, generally at about 180 DEG C to about 200 DEG C Under carry out about 5 to about 15 minutes Grape berry (see for example, Schmidt and Thomsen, Biores.Technol., 64: 139-151[1998]；Palonen et al., Appl.Biochem.Biotechnol., 117:1-17 [2004]；Varga et al., Biotechnol.Bioeng.,88:567-574[2004]；Martin et al., J.Chem.Technol.Biotechnol., 81: 1669-1677[2006]).This pretreatment preferably at dry, the dry of about 2 to about 30% of about 1% to about 40%, or about 5 Carry out under dry to about 20%, and generally original ph raises because adding alkali such as sodium carbonate.In some embodiments In, a kind of amendment to wet oxidation method for pretreating of referred to as wet method explosion (wet oxidation and the combination of steam explosion) can be used Scheme.This method can process the dry of the most about 30%.In wet method explosion, during pretreatment when specific stop Oxidant is introduced after between.Then pretreatment (see for example, WO2006/032282) is terminated by flash distillation to atmospheric pressure.

In some embodiments, ammonia filament expansion (AFEX) can be used.The method is included in moderate temperature e.g., from about 90 Cellulosic material is processed with liquid or gaseous ammonia under to about 100 DEG C and high pressure e.g., from about 17 to about 20 bars, the most about 5 to about 10 points Clock, wherein dry matter content may be up to about 60% (see for example, Gollapalli et al., Appl.Biochem.Biotechnol.,98:23-35[2002]；Chundawat et al., Biotechnol.Bioeng., 96: 219-231[2007]；Alizadeh et al., Appl.Biochem.Biotechnol., 121:1133-1141 [2005]；With Teymouri et al., Biores.Technol., 96:2014-2018 [2005]).AFEX pretreatment cause cellulose depolymerization and The partial hydrolysis of hemicellulose.Lignin-carbohydrate compound is cleaved.Dilute ammonia pretreatment make use of diluter than AFEX Ammonia solution, and can carry out at the temperature of about 100 DEG C to about 150 DEG C or the most any suitable temperature (see for example, beautiful State's public announcement of a patent application the 2007/0031918th and 2007/0037259, is incorporated by herein by quoting).At some In embodiment, the persistent period of dilute ammonia pretreatment is about 1 to about 20 minute, or the most any suitable persistent period.

In some embodiments, organic solvent pretreatment can be used.This method is by about 160 DEG C to about 200 DEG C Under, with ethanol water (ethanol of about 40% to about 60%) extraction cellulosic material was taken off in about 30 to about 60 minutes wooden (see, Such as Pan et al., Biotechnol.Bioeng., 90:473-481 [2005]；Pan et al., Biotechnol.Bioeng., 94:851-861[2006]；And Kurabi et al., Appl.Biochem.Biotechnol., 121:219-230 [2005]). It is usually added into sulphuric acid as catalyst.In organic solvent pretreatment, major part hemicellulose is removed.

The example of other suitable preprocess methods be known in the art (see for example, Schell et al., Appl.Biochem.Biotechnol.,105:69-85[2003]；Mosier et al., Biores.Technol., 96:673- 686[2005]；And U.S. Patent Application Publication the 2002/0164730th).

In some embodiments, Chemical Pretreatment is preferably carried out with acid treatment, and more preferably with continuous print diluted acid Process and/or weak acid treatment.Acid is typically sulphuric acid, but is used as other acid, such as nitric acid, phosphoric acid, hydrochloric acid or its mixing Thing.Weak acid treatment about 1 to about 5 about 1 to about 4 or about 1 to about 3 pH value range in carry out.In some embodiments, Acid concentration is in the scope of the acid from about 0.01 to about 20wt%, and in other embodiments, its from about 0.05 to about In the scope of the acid of 10wt%, in other embodiments, it is in the scope of the acid from about 0.1 to about 5wt%, and again at other In embodiment, it is in the scope of the acid from about 0.2 to about 2.0wt%.Described acid is made to contact and at holding with cellulosic material In preferably about 160 DEG C to about 220 DEG C, and at a temperature in the range of the most about 165 DEG C to about 195 DEG C, continue from several seconds Time (such as, about 1 second to about 60 minutes) to a few minutes.

In some embodiments, pretreatment is carried out in aqueous slurry.In some embodiments, deposit during pretreatment Between preferably about 10 to about 80wt% or about 20 to about 70wt% or about 30 to about 60wt% or the fiber of amount of about 50wt% Cellulosic material.The cellulosic material of pretreatment can not be washed or use any suitable method known in the art washing (such as, to use Water washs).

Physics pretreatment.Physics pretreatment can include high pressure and/or high temperature (steam explosion).In some embodiments, High-pressure physics pretreatment includes in the range of about 300 to about 600psi or about 350 to about 550psi or about 400 to about 500psi Pressure or the pressure of about 450psi.In some other embodiments, high temperature pretreatment includes using about 100 DEG C to about 300 DEG C or about 140 DEG C to about 235 DEG C in the range for the treatment of temperature.In some embodiments, mechanical pretreatment is with batch process (such as, Sunds hydrolyzer is carried out in using the steam gun hydrolyzer system of high pressure as defined above and high temperature；Sunds Defibrator AB,Sweden)。

United physics and Chemical Pretreatment.In some embodiments, united physics and Chemical Pretreatment can be used. It is true that can physically and chemically pre-treating cellulosic material.Such as, in some embodiments, pre-treatment step includes Diluted acid or weak acid treatment and high temperature and/or HIGH PRESSURE TREATMENT.Schedule if desired, physics and Chemical Pretreatment can sequentially or simultaneously be entered OK.In some other embodiments, mechanical pretreatment can also be used in combination with physics and Chemical Pretreatment.Therefore, exist In some embodiments, cellulosic material is carried out machinery, chemically or physically pretreatment, or its any combination, to promote fiber Element, hemicellulose and/or the separation of lignin and/or release.

Biological Pretreatment.In some embodiments, Biological Pretreatment Techniques can be used.In some embodiments, this A little methods include applying solubilising lignin microorganism (see for example, Hsu, inWyman (edit),Handbook on Bioethanol:Production and Utilization, Taylor &Francis, Washington, D.C., 179-212 page [1996]；Ghosh and Singh, Adv.Appl.Microbiol., 39:295-333 [1993]；McMillan, Baker and Overend (edits),Enzymatic Conversion of Biomass for Fuels Production,ACS Symposium Series566, American Chemical Society, Washington, D.C., the 15 is in chapter [1994]； Gong et al., Adv.Biochem.Engineer.Biotechnol., 65:207-241 [1999]；Olsson and Hahn- Hagerdal,Enz.Microb.Tech.,18:312-331[1996]；And Vallander and Eriksson, Adv.Biochem.Eng.Biotechnol.42:63-95[1990])。

In some embodiments, from solid, separate the soluble compound obtained from pretreating process subsequently.Such as, In some embodiments, separating step includes mechanical means (such as, screening, sieving, the centrifugal or mistake of one or more standards Filter) to realize separating.In some other embodiments, from solid, after pretreatment, do not separate soluble compound.This area Artisans understand that, pretreatment can as in batches, fed-batch or continuous processing carry out.Also will should be understood that Schedule if desired, Pretreatment can low, in or high solid denseness under carry out that (see for example, WO2010/022511, it is incorporated to by reference of text Herein).

Fermentation

In some embodiments, the method for producing sugar as herein described also includes becoming eventually obtained sugar fermentation Product.Fermentation includes by using fermenting organisms that sugar source is changed into end-product.Any suitable organism can be used on this In bright, including being suitable for producing the antibacterial of desired end-product and fungal organism (such as, yeast and filamentous fungi).Especially Its suitable fermenting organisms can be by sugar, and such as glucose, fructose, maltose, xylose, mannose and/or arabinose are straight Connect or fermentation indirectly (that is, converting) becomes desired end-product.The example of fermenting organisms includes fungal organism such as yeast. In some embodiments, yeast strain can be used, include but not limited to lower dependent of dead military hero: belong to Saccharomyces (such as, saccharomyces cerevisiae (S.cerevisiae) and Saccharomyces uvarum (S.uvarum))；Pichia (such as, pichia stipitis (P.stipitis) and pichia pastoris phaff (P.pastoris))；Mycocandida (such as, Candida utilis (C.utilis), C.arabinofermentans, Di Dansi candida mycoderma (C.diddensii), C.sonorensis, Xiu Ha Tower candida mycoderma (C.shehatae), candida tropicalis (C.tropicalis) and Candida boidinii (C.boidinii)).Other fermenting organisms include but not limited to zymomonas, Hansenula (such as, multiform Hansen Yeast (H.polymorpha) and Hansenula anomala (H.anomala), Kluyveromyces (such as, Kluyveromyces fragilis ) and Schizosaccharomyces (such as, the bacterial strain of schizosaccharomyces pombe (S.pombe) (K.fragilis).

In some embodiments, fermenting organisms is Escherichia (Escherichia) (such as, escherichia coli (E.coli)), zymomonas (such as, zymomonas mobilis (Z.mobilis)), fermentation Bacillus (Zymobacter) (such as, Z.palmae), Klebsiella (Klebsiella) (such as, acid-producing Klebsiella bacterium (K.oxytoca)), bright string Pearl Pseudomonas (Leuconostoc) (such as, Leuconostoc mesenteroides (L.mesenteroides)), fusobacterium (such as, aminobutyric acid Clostridium (C.butyricum)), Enterobacter (Enterobacter) (such as, clostridium perfringen (E.aerogenes)) and heat detests Oxygen Bacillus (Thermoanaerobacter) (such as, hot anaerobic bacillus(cillus anaerobicus) belong to BG1L1 [see for example, GeorgievaheAhring, Appl.Microbiol, Biotech., 77:61-86], the hot anaerobic bacillus(cillus anaerobicus) of producing and ethanol (T.ethanolicus), it is pyrolyzed sugared hot anaerobic bacillus(cillus anaerobicus) (T.thermosaccharolyticum) or the hot anaerobic bacillus(cillus anaerobicus) in Maxwell (T.mathranii)), Lactobacillus (Lactobacillus), Corynebacterium glutamicum R (Corynebacterium Glutamicum R), hot glucosidase bacillus cereus (Bacillus thermoglucosidaisius) and hot glucosidase ground The bacterial strain of bacillus cereus (Geobacillus thermoglucosidasius).It is specific that expection fermenting organisms is not limited to these Bacterial strain because any suitable organism can be with in the present invention.

Fermentation condition depends on desired tunning and can be readily determined by those of ordinary skill in the art.Relating to By in some embodiments of culture propagation ethanol, fermentation typically lasts for about 1 hour to about 120 hours or about 12 to about 96 Between hour.In some embodiments, fermentation is between about 20 DEG C to about 40 DEG C or between about 26 DEG C to about 34 DEG C or about Carry out at a temperature of 32 DEG C.In some embodiments, the pH of fermentation be about pH3 to about pH7, and in some other embodiments In, pH is about 4 to about 6.

In some embodiments, enzyme hydrolysis and fermentation are carried out, so that each biological respinse can in separate container Under its respective optimum condition, (such as temperature) is carried out.In some other embodiments, with simultaneous saccharification and fermentation (i.e. " SSF ") reaction, the method producing glucose from cellulose is carried out with fermentation simultaneously.In some embodiments, SSF generally exists Carrying out at a temperature of about 28 DEG C to about 50 DEG C or about 30 DEG C to about 40 DEG C or about 35 DEG C to about 38 DEG C, this is to most of fibers Element enzymatic mixture be optimal about 50 DEG C and be optimal about 28 DEG C to most of yeast between about 30 DEG C compromise.But, Expection the invention is not restricted to any specific temperature, because any suitable temperature can be with in the present invention.

In some embodiments, the method being used for producing glucose also includes that glucose fermentation becomes desired to be produced eventually Thing.Expect that method provided herein is not limited to the generation of any specific end-product.In some embodiments, end-product includes Fuel alcohol or precursor industrial chemical.Such as, in some embodiments, tunning includes precursor industrial chemical, such as Alcohol (such as, ethanol, methanol and/or butanol), organic acid (such as, butanoic acid, citric acid, acetic acid, itaconic acid, lactic acid and/or glucose Acid), ketone (such as, acetone)；Aminoacid (such as glutamic acid)；Gas (such as, H2 and/or CO2)；Antimicrobial is (such as blue or green Mycin and/or tetracycline)；Enzyme；Vitamin (such as, riboflavin, B12 and/or beta-carotene) and/or hormone.Real at some Executing in scheme, end-product is fuel alcohol.Suitably fuel alcohol is as known in the art and includes, but are not limited to lower alcohol such as first Alcohol, ethanol, butanol and propanol.

The water-disintegrable expression of enzymes of sugar increases

In embodiments more provided herein, fungal cell is also sugared to one or more to increase it by genetic modification The production of water-disintegrable enzyme.Such as, in some embodiments, fungal cell's process LAN coding glycosylhydrolase such as β-glucosyl enzym Homology or heterologous gene.In some embodiments, one or more glycosylhydrolases described are cellulase as herein described. Such as, in some embodiments, described enzyme is any one of following multiple enzyme: endoglucanase, cellobiose water Solution enzyme, β-glucosyl enzym, endoxylanase, xylobiase, arabinofuranosidase, α-glucuronidase, acetyl wood gather Sugar ester enzyme, Resina Ferulae acyl esterase and α-glucuronyl-esterase and/or the enzyme of any other involved in sugar hydrolysis.In some embodiments In, fungal cell by genetic modification with increase β-glucosyl enzym expression.Therefore, in some embodiments, fungal cell's bag Polynucleotide sequence containing the expression of the polynucleotide for increasing coding β-glucosyl enzym.In some embodiments, fungus Cell is also encoded the polynucleotide of one or more endogenous cellobiose dehydrogenases by genetic modification with disappearance.

In some embodiments, the water-disintegrable enzyme of described sugar is endogenic for described fungal cell, and real at other Executing in scheme, the water-disintegrable enzyme of described sugar is ectogenic for fungal cell.In some other embodiments, enzymatic mixture Also comprise the water-disintegrable enzyme of sugar that described fungal cell is heterologous.Further, in some embodiments, it is used for producing The method of glucose includes making cellulose and comprises the enzymatic mixture to the water-disintegrable enzyme of sugar that described fungal cell is heterologous and connect Touch.

In some embodiments, any one of multiple suitable method well known by persons skilled in the art side is used Method by fungal cell's genetic modification with increase glycosylhydrolase expression.In some embodiments, the multinuclear of water-disintegrable enzyme is encoded Nucleotide sequence is suitable in host fungal cell for increasing expression.As used herein, the many nucleoside expressed it are suitable for Acid sequence is to be inserted into expression vector or the polynucleotide that are otherwise modified to comprise in host cell express institute Required controlling element, the polynucleotide sequence placed in the way of allowing these polynucleotide to express in this host cell.Table Reach these required controlling elements and include promoter sequence, transcriptional initiation sequence and optionally, enhancer sequence.Such as, In some embodiments, polynucleotide sequence is inserted into the plasmid vector being suitable in fungal host cells expressing In.

Experiment

Following example describe the present invention in further detail, and described embodiment is intended to limit never in any form wanted Seek the scope of the present invention of protection.

In following experiment disclosure, apply following abbreviation: ppm (million/)；M (mol/L)；MM is (in the least Mol/L), uM and μM (micromoles per liter)；NM (nanomole/liter)；Mol (mole)；Gm and g (gram)；Mg (milligram)；Ug and μ g (microgram)；L and l (rises)；Ml and mL (milliliter)；Cm (centimetre)；Mm (millimeter)；Um and μm (micron)；Sec. (second)；Min (point Clock)；H (hour)；U (unit)；MW (molecular weight)；Rpm (revolution per minute)；DEG C (degree Celsius)；Wt% (percentage by weight)； W.r.t. (about)；DNA (DNA (deoxyribonucleic acid))；RNA (ribonucleic acid)；HPLC (high pressure liquid chromatography)；MS (mass spectrum)；LC (liquid chromatograph)；LC/MS (liquid chromatography/mass spectrometry)；LC/MS/MS (liquid chromatograph/multi-stage ms)；HMF (Hydroxymethylfurfural)； YPD (yeast extract 10g/L；Peptone 20g/L；Glucose 20g/L)；DCPIP (2,6-chlorophenesic acid indole phenol)；CV (column volume)；NREL (National Renewable Energy laboratory, Golden, CO)；ARS (ARS culture collection center or NRRL Culture collection center, Peoria, IL)；Lallemand(Lallemand Ethanol Technology,Milwaukee, WI)；Cayla(Cayla-InvivoGen,Toulouse,France)；Agilent New Brunswick(New Brunswick Scientific Co.,Edison,NJ)；Sigma(Sigma Aldrich,St.Louis,MO)； Eppendorf(Eppendorf AG,Hamburg,Germany)；GE Healthcare(GE Healthcare,Waukesha, WI)；Bruker Optics(Bruker Optics,Inc.,Billerica,MA)；Specac(Specac,Inc., Cranston,RI)；Invitrogen(Invitrogen,Corp.,Carlsbad,CA)；Alphalyse(Alphalyse, Inc.,Palo Alto,CA)；Promega(Promega,Corp.,Madison,WI)；Sartorius(Sartorius- Stedim Biotech,SA,Aubagne,France)；Finnzymes(Finnzymes Oy,Espoo,FI[Thermo The part of Fisher Scientific]), CalBiochem (CalBiochem, EMD Chemicals, Inc., Gibbstown, NJ)；With Bio-Rad (Bio-RadLaboratories, Hercules, CA).

Following CDH sequence from thermophilic fungus destroyed wire (C1) can be with in the present invention.SEQ ID NO:1 and 2 provides respectively The nucleotide sequence of CDH1 and aminoacid sequence.SEQ ID NO:3 is the aminoacid sequence of CDH2, and SEQ ID NO:4 is CDH3 Aminoacid sequence, SEQ ID NO:5 is the aminoacid sequence of CDH4, and SEQ ID NO:6 is the aminoacid sequence of CDH5, SEQ ID NO:7 is the aminoacid sequence of CDH6, and SEQ ID NO:8 is the aminoacid sequence of CDH7.

CDHI

atgaggacctcctctcgttaatcggtgcccttgcggcggcactcttgccgtctgcccttgcgcagaacaacgcgccg gtaaccttcaccgacccg gactcgggcattaccttcaacacgtggggtctcgccgaggattctccccagactaagg gcggtttcacttttggtgttgctctgccctctgatgccctcacgacagacgccaaggagttcatcggttacttgaaa tgcgcgaggaacgatgagagcggttggtgcggtgtctccctgggcggccccatgaccaactcgctcctcatcgcggc ctggccccacgaggacaccgtctacacctctctccgcttcgccaccggctatgccatgccggatgtctaccaggggg acgccgagatcacccaggtctcctcctctgtcaactcgacgcacttcagcctcatcttcaggtgcgagaactgcctg caatggagtcaaagcggcgccaccggcggtgcctccacctcgaacggcgtgttggtcctcggctgggtccaggcatt cgccgaccccggcaacccgacctgccccgaccagatcaccctcgagcagcacgacaacggcatgggtatctggggtg cccagctcaactccgacgccgccagcccgtcctacaccgagtgggccgcccaggccaccaagaccgtcacgggtgac tgcggcggtcccaccgagacctctgtcgtcggtgtccccgttcctacgggcgtctcgttcgattacatcgtcgtggg cggcggtgccggtggcatccccgccgccgacaagctcagcgaggccggcaagagtgtgctgctcatcgagaagggct ttgcctcgaccgccaacaccggaggcactctcggccccgagtggctcgagggccacgaccttacccgctttgacgtg ccgggtctgtgcaaccagatctgggttgactccaaggggatcgcttgcgaggataccgaccagatggctggctgtgt cctcggcggcggtaccgccgtgaatgccggcctgtggttcaagccctactcgctcgactgggactacctcttcccta gtggttggaagtacaaagacgtccagccggccatcaaccgcgccctctcgcgcatcccgggcaccgatgctccctcg accgacggcaagcgctactaccaacagggcttcgacgtcctctccaagggcctggccggcggcggctggacctcggt cacggccaataacgcgccagacaagaagaaccgcaccttctcccatgcccccttcatgttcgccggcggcgagcgca acggcccgctgggcacctacttccagaccgccaagaagcgcagcaacttcaagctctggctcaacacgtcggtcaag cgcgtcatccgccagggcggccacatcaccggcgtcgaggtcgagccgttccgcgacggcggttaccaaggcatcgt ccccgtcaccaaggttacgggccgcgtcatcctctctgccggtacctttggcagtgcaaagatcctgctgaggagcg gtatcggtccgaacgatcagctgcaggttctcgcggcctcggagaaggatggccctaccatgatcagcaactcgtcc tggatcaacctgcctgtcggctacaacctggatgaccacctcaacaccgacactgtcatctcccaccccgacgtcgt gttctacgacttctacgacccgtgggacaatcccatccagtctgacaaggacagctacctcaactcgcgcacgggca tcctctcccaagccgctcccaacattgggcctatgttctgggaagagatcaagggtgcggacggcattgttcgccag ctccagtggactgcccgtgtcgagggcagcctgggtgcccccaacggcaagaccatgaccatgtcgcagtacctcgg tcgtggtgccacctcgcgcggccgcatgaccatcaccccgtccctgacaactgtcgtctcggacgtgccctacctca aggaccccaacgacaaggaggccgtcatccagggcatcatcaacctgcagaacgccctcaagaacgtcgccaccctg acctggctcttccccaactcgaccatcacgccgcgccaatacgttgacagcatggtcgtctccccgagcaaccggcg ctccaaccactggatgggcaccaacaatctcggcaccgacgacgggcgcaagggcggctccgccgtcgtcgacctca acaccaaggtctacggcaccgacaacctcttcgtcctcgacgcctccatcttccccggcgtgcccaccaccaacccc acctcgtacatcgtgacggcgtcggagcacgcctcggcccgcatcctcgccctgcccgacctcacgcccgrccccaa gtacgggcatgtcggcggccgcgaatggagcggcagcttcgtctgcgccgacggctccacgtgccagatgcagaacg agtggtcctcgcagtgcttgtga(SEQID NO:1)

MRTSSRLIC]ALAAALLPSALAQNNAPVTFTDPDSCrlTFNTWC]LAEDSPQTKC]C] FTFCiVALPSDALTTDAKEFICJYLKC.ARNDESt@UlCtJVSLt@t@PMTNSLLIAAWPHEDTVYTSLRFATC] YAMPDVYQCJDAEITQVSSSVNSTHFSLIFRCENC.LQWSQSC]ATCJC@ASTSNCJVLVLCJ\ ITVQAFADPCINPTCPDQITLEQHDNC]MCJIWClAQLNSDAASPSYTEWAAQATKTVTClDCCJCJPTETSVVCJV PVPTCIVSFDYIVVC]ClCTACJCJIPAADKLSEACJKSVLLIEKC]FASTANTCIC] TLCTPEWLECIHDLTRFDVPC]LC.NQIWVDSKCJIACEDTDQMACJCVLCJCICJTAVNACILWFKPYSLDWDYLF PSCJUIKYKDVQPAINRALSRIPCJTDAPSTDCJKRYYQQCIFDVLSKCJLACJCJCJWTSVTANNAPDKKNRTFSH APFMFACJC@ERNCJPLCJTYFQTAKKRSNFKLWLNTSVKRVIRQCJCrHITCVEVEPFRDCJCJYQt@ iVPVTKVTCJRVILSACJTFCJSAKILLRSCJICJPNDQLQVVAASEKDCJPTMISNSSWINLPVCJYNLDDHLNTD TVISHPDVVFYDFYEAWDNPIQSDKDSYLNSRTCJILAQAAPNICTPMFWEEIKCJADCJIVRQLQWTARVECJSLC JAPNCJKTMTMSQYLCIRCJATSRCIRMTITPSLTTVVSDVPYLKDPNDKEAVIQt]IINLQNALKNVANLTWLFPN STITPRQYVDSMWSPSNRRSNHWMC@TNKICITDDCJRKCICISAVVDLNTKVYCJTDNLFVIDASIFPCrVPTTNP TSYIVTASEHASARILALPDLTPVPKYC@QCCrCrREWSCrSFVCADCrSTCOMONEWYSQCL(SEQIDNO:2)

CDH2.

MKLLSRVCJATALAATLSLQQC'AAQMTECJTYTDEATCJIQFKTWTASECJAPFTFCrLTLPADALEK DATEYICJLLRCQITDPASPSWCCJISHCTQSCJQMTQALLLVAWASEDTVYTSFRYATCJYTLPt@ LYTtJDAXLTQISSSVSEDSFEVLFRCENCFSWDQDCJTKCJNVSTSNCJNLVLCJRAAAKDCJVTCJPTCPDTAEF CIFHDNCIFC!QWCJAVLECIATSDSYEEWAKLATTTPETTCDCJTCJPCJDKECVPAPEDTYDYIVVCJACJACJC iITVADKLSEACJHKVLLIEKCJPPSTCrLWNCJTMKPEWLESTDLTRFDVPCJLCNQIWVDSACJTAC.TDTDQMA CiCVLCiCiCJTAVNACrLUTWKPHPADWDENFPECrWKSSDLADATERVFKRIPCITSHPSQDCJKLYRQECJFEV ISKCJLANAClWKEISANEAPSEKNHTYAHTEFMFSCJCiERCrCtPLATYLASAAERSNFNLWLNTAVRRAVRSC@ SKVTCJVELECLTDC]CJFSCiTVNLNECrCJCJVIFSACrAFCJSAKLLLRSCJICJPEDQLEIVASSKDCiETFT PKDEWINLPVCiHNLIDHLNTDLIITHPDVVFYDFYAAWDEPITEDKEAYLNSRSCrILAQAAPNICJPMMWDQVTP SDCJITRQFQWTCRVECJDSSKTNSTHAMTLSQYLC@RC]VVSRCJRMCJITSCJLSTTVAEHPYLHNNCJDLEAVI Qt@IQNVVDALSQVADLEUTVLPPPDC@TVADYVNSLIVSPANRRANHWMCJTAKLCJTDDC@RSCIt@ TSVVDLDTKVYCrTDNLFVVDASVFPCiMSTCJNP5AMIVIVAEQAAQRILALRS(SEQIDNO:3)

C'DH3.

MKFLRKSDRCiSVLCrSTLFSLAFLFYSPPTAAQSPPPDCiAVYDYIVICiSCiPCJCiCJVVCiANLAKACiYSVL LLEACrDDSPCrACJFC]VYTPTVTWDFYVKHYPECiDPRDNQYSHLTU'LTPDC] RYWVCJQSCIAPECISRLLCJVYYPRCIATLCIC]SSMINAMVVWLPNDSDWDYHAEVTCJDDSWRAENMHKlFQKI EKNNYLPRCJTANHCrFDCrUrFQTQMCrTMVQTNRTCJPLQC]NCiVMTTYAQDU' NLTIPMSDLLIRDPNEICiPDRDQTSSIYCiQVSHQFANCrNRYSSRHYVQDAVSSCJANLTVSLTSLATRILFDTV TEPDSPRATCIVEYLFCIKSLYRClDRRRADCJAICIVNRTAVARREV[VSCICIAFNSPQLLLLSCJIClNATELE ALCilPVIRDLPCiVC]RNLMDNQEMPIVCiTCiSPCiCiCrPCiAVACiVAMYKTRHPAHCrERDMFLFCICIPC] FLFRCIFWPNEAVHLPDEPAQPVYCIVSMVKCISSVNNCICIU'VKLRSRDPTDTPEINFNHYAVCJAEYDLEAVKD TVAUTIRSVYRRVCJIATVEPFCARCIPDENCIYCClEEDEAWIHKQTFCiHHPTSTNKICrADDDPTAVLDSKFRV RCiVRALRVVDASAFARIPCJVFPVVSTFMISQKASDDILAELEAESR(SEQIDNO:L1)

C'.DH4.

MCIFLAATLVSCAALASAASIPRPHAKRQVSQLRDDYDFVIVClCICJTSCILTVADRLTEAFPAKNVLVIEYCIDV HYAPCITFDPPTDW]TPQPDAPPSUTSFNSLPNPDMANTTAFVLACIQVVC]C1SSAVNC@ MFFDRASRHDYDAUTTAVC@CrSC@FEQSSHK\hiDUTECJLFPFFQKSVTFTEPPADIVQKYHYTWDLSAYCiNCr STPIYSSYPVFQWADQPLLNQA\ITQEMCilNPVTECAC@CrDKECiVCWVPASQHPVTARRSHAC@ LCJHYADVLPRANYDLLVQHQVVRVVFPNC@PSHCIPPLVEARSLADNHLFNVTVKCrfVIISAC@ ALHTPTVLQRSC@It@PASFLDOACJIPVTLDLPCJVC@ANLQDHCt@PPVTUTNYTEPYTCJFFPLPSEMVNNATF KAEAITCIFDEVPARCIPYTLACJCINNAIFVSLPHLTADYC@AITANIRAMVADCJTAASYLAADVRTIFt@ MYAC@YEAQLLVLADLLDNPEAPSLETPU'ATSEAPQTSSVLAFLLHPLSRCISVRLNLSDPLAQPVLDYRSC] SNPVDIDLHLAHVRFLRCJLLDTFTMQARCJALETAPt@SAVADSDEALC@EYVRSHSTLSFMHPCCTAAMLPEDRC @CrVVt@PDLKVHCiAEC@LRVVDMSVMPLLPCIAHLSATAYAVCJEKAADIIIQEWMDKEQ(SEQIDNO:5)

CDH5.

MELLRVSLAAVALSPLILFCJVAAAHPTARSIARSTILDCJADCJLLPEYDYI11CrCJCJTSCrLTVADRLTENRK RKFSRSPLPTSPARSSPAWCYSVLVLERClIFQNSSSVTTISCJCrSRCrLFDPSLTFNINSVPOACJLDNRSIAVI CJCiLILCiCiSSCJVNCrLQVLRCrOREDYDRWt@SYFtiPNSDWSU'KCJLLPYFKKAWNFHPPRPELVSOFDIK YDPSY\IIC:NTSDVHASFPTTFWPVLKLEMAAFCJDIPCJVEYPPDSASCJETCJAYU' HPASVDPATVLRSFARPAHWDNIEAARPNYHTLTCJQRVLKVAFDCJNRATSVVFVPANATDHSTARSVKAKKEIVL AACJAIHTPQILQASCTVCJPKQVLKEAC@VPLVVDAPCIVCJSNFODQPYVVAPTFNFTKFPFHPDFYDMILNQTF IAEAQAOFEKDRTClPHTIASCYCCJSWLPLQIIAPNSWKDIARRYESQDPAAYLPACJTDETVIECJYRAQQKALA RSMRSKQSAMYNFFLRCJCJYEECISVVYLHPTSRCITVRINRSDPFFSPPEVDYRALSNPTDLEVLLEFTPFTRRY FLETRLKSLDPVELSPCJANVTAPADIEAWLRSVMIPSSFHPTCJTAAIV1LPRHLCrCJVVDENLLVYCiVECiLS VVDASVMPDLPClSYTQQTVYAIAEKAADLIKSRA(SEQIDNO:C))

C'.DHCl'

MQVASKLVAVTCJCrALALvliLHPVAAQECiCTNISSTETYDYIVVC]SCrACtC]IP@ iADRLSEACrHKVLLIEKCJPPSTC@RUTC@t@iMKPEWLiCiTNLTRFDVPt@LC.NQIWADPTCrAICTDVDQMA CJCMLC@CJCITAVNAC.LW\/IIKpHPADWDVNFPECIUTHSEDMAEATERVFERIPCJTITPSMDClKRYLSOCJ FDMLCrCJSLEAACrWEYLVPNEHPDRKNRTYCJHSTFMYSCJCJERCJCJPLATYLVSAVQRECrFTLUTMNTTVT RIIRECTCJHATCJVEVQCSNSEACJQACJIVPLTPKTCJRVIVSACJAFCJSAKLLFRSClICJPKDQLN [VKNSTDCJPSMISEDQUTIELPVCiYNLNDHVClTDIEIAHPDVVFYDYYCJAUTDEPIVEDTERYVANRTCiPLA QAAPNICiPIFWETIKCiSDCiVSRHLQWQAR@rECiXLNTSMTITQYLCiTCiSRSRClRMTITRRLNTVVSTPPY LRDEYDREAVIQC]IANLRESLKCIVANLTUTITPPSNVTVEDFVDSIPATPARRCSNHWICJTAKIClLDDC1REC lCTSVVDLNTKVYCTDNIFVVDASIFPClHITClNPSAAIVIAAEYAAAKILALPAPEDAAS(SEQIDNO:7)

CDH7

MASVDLDQPFDYIVVCrCrCrTACiLVVANRLSEDSMVRVLVVEACJADRNADPLVLTPCJLVACJLYCiKDEYDUT NFSSPPQPTLNNRRINQARC@KMLCJCJTSC]LNFMMLLYPSKC@NIDSWAALC] NPSWNYDALAPYLRKFATVHPSPQSARDLLCJLTYIDESLAAC]DC]PIQVSHTDCJHNVTNKAUTLETFASLC@ LEVSTDPRDC@KALCrAFQNHASIDPATHTRSFACJPAYYTPDVAKRPNLVVLTETLVARhlLFDTACiCiECJDAV ATCiVEllTKDCiQKKQVSACCiEVILAACrALQSPQlLELSCJVC@CiRELLEKHNIPVVVDNPNVC@ EHVQDHPlVC.QSFEVADCJVPSCiDVLRDPNVLQAVVCJMYQSCJCrCJAC@ PLCJQSVISVAYTPLVDCJSCIVVSAEAKAELLARHESSFSTAECJKVLRDLVESPSEATFEFLLFPSQVDlPENPT SMAQYITPVLPENYISVMTFIHQPFSRCJKVHITSPDIRAAPLWDPRYNSDPLDLELLARCJVQFVERIVDSATPFC rRVLKQCJCJKRQPPLRADDLETAREIVRQRQISVFHVSCiSCTMRPRDQCrCJVVDERLRVYCJTRCJLRVVDASV FPIEPVCiNIQSVVYAVAERAADLIKEDRAKA(SEQIDNO:g)

Embodiment 1

Fungal bacterial strain and method

This embodiment describes the generation of the variant of fungal bacterial strain C1.

Strain Designation

Bacterial strain CF-200 (UV18#100f Δ alpl) is derivative C1 bacterial strain.Bacterial strain CF-400 is the derivant of C1 bacterial strain (" UV18#100f Δ alpl Δ pyr5 "), is further embellished by disappearance cdh1,

Wherein cdh1 comprises polynucleotide sequence SEQ ID NO:1.Cellulolytic enzyme from these bacterial strains passes through Method well known in the art and suitable fungus growth medium is used to be produced by deep-layer liquid cultivation and fermentation.

GOPOD measures

GOPOD measures test kit (Sigma-Aldrich) and is used for measuring in these experiments the amount of produced glucose. In these experiments, the GOPOD that the given the test agent of 10ul adds to 190ul to be provided by this test kit measures in mixture.Allow Reactant vibrates 30 minutes at 50 DEG C.Measure solution absorbance at 510nm to determine the amount of produced glucose.Calculate The concentration of glucose of these samples compared with dextrose standard sample (0-150 grams per liter).

Embodiment 2

The purification of C1CDH1

In this embodiment, the 400mL C1 supernatant that the method using embodiment 1 is prepared by rotary evaporator is first used It is concentrated into 140mL.Then, 4 online Hi-Prep26/10 desalting column (in-line Hi-Prep26/10desalting are used Column, GE Healthcare, 17-5087-02) by 63mL concentrated solution buffer-exchanged to 20mM MOPS pH of buffer 7.0 In.It is loaded to comprise with 20mM pH7.0MOPS buffer by the supernatant (～150g/L total protein) of the buffer-exchanged of gained The post of 500mL DEAE Fast Flow resin (GE Healthcare, 17-0709-01) of pre-balance.Use 1 column volume (CV) 20mM MOPS (pH7.0) cleans post, then runs 0-300mM NaCl gradient through 12 column volumes.Collect fraction also ByBis-tris PAGE gel (Invitrogen, NP0322BOX) is analyzed.Corresponding CDH1 Apparent molecular weight SDS-PAGE band by MS analyze (being carried out by Alphalyse).Quality Map analysis of spectrum confirms that the later stage washes The existence of CDH1 in de-fraction.Collect the fraction comprising CDH1 as proved by PAGE gel and confirmed by MS, and lead to Cross use Sartorius centrifuge 10kDa filter (Sartorius-Stedim, VS2002) ultrafiltration to concentrate.Then, will 10mL500mM piperazine (pH5.6) and 45mL saturated ammonium sulfate join the thing that collects comprising CDH1 of 45mL, and mixing gained Compound is loaded to use 50mM piperazine, Phenyl FF (high sub) 16/10 post of the 1.6M ammonium sulfate pre-balance in pH5.6 (GE Healthcare,28-9365-45).50mM piperazine, in pH5.6, the gradient of 1.6M to 0M ammonium sulfate runs through 30CV. Collect fraction and as described above selected fraction carried out PAGE gel analysis, being disclosed in eluting in last cleaning step CDH1 there is about 80-90% purity.

CDH1 activity uses to be similar to and (is seen, Schou et al., Biochem J., 330:565-71 by Schou et al. [1998]) the DCPIP reduction inspection described in is measured.In flat 96 orifice plates of saturating ultraviolet light, 50 μ L are comprised the fraction of CDH1 It is added to 1.0g/L cellobiose and 100 μMs of DCPIP in 100mM sodium acetate, 150 μ L solution in pH5.0.Of short duration in room temperature Ground agitation sample, then measures the absorbance (A at 530nm₅₃₀) 10 minutes.The fraction comprising C1CDH1 is illustrated in the suction of 530nm The rapid decrease of luminosity.DCPIP inspection uses the CDH1 of different amounts of glucose or cellobiose and purification to carry out.Shallow in 96 holes Orifice plate prepares cellobiose (1.0g/L to 7.8mg/L) and the serial dilution thing of glucose (10g/L to 78mg/L).Then, 20 μ L glucose and cellobiose standard substance are added to 160 μ L/ hole 200mM DCPIP (in 100mM pH5.0 sodium acetate).Instead Should start by adding 20 μ L CDH1 solution.Monitor the absorbance 30 minutes at 530nm.Minimizing at the absorbance of 530nm Relatively showing of speed, C1CDH1 to the activity of cellobiose comparison glucose larger about 10 times.

Embodiment 3

Cdh1 isolates the preparation of mark deletion construct

Genomic DNA uses standardization program from C1 strains separation.Briefly, mycelium inoculation thing is inoculated into grown cultures In base and allow 35 DEG C grow 72 hours.Centrifugal mycelium block of collecting, washing, and add 50 μ L DNA extraction buffer (200mM TRIS,pH8.0；250mMNaCl；125mM EDTA；0.5%SDS).Mycelium taper dismembyator is ground, uses 250 μ L Extraction buffers extract again, and centrifugal suspension.Supernatant is transferred in the new test tube comprising 300 μ L isopropanols.From The heart collects DNA, by 70% washing with alcohol twice, and dilution in 100 μ L water.

The genomic DNA fragment of cdh1 gene flank use primer cf09067 and cf09068 (cdh1 upstream homologous) with Primer cf09069 and cf09070 (cdh1 downstream homology) clones.PCR reaction is by usingPolymerase (Promega) according to the manufacturer's instructions and use every kind of primer of 0.2 μM to carry out.Amplification condition be 95 DEG C 2 minutes, be followed by 95 DEG C continue to continue for 30 seconds, 55 DEG C 30 seconds (for upstream homologous) or 53 DEG C continue 30 seconds (for downstream homology), 72 DEG C Continue 35 circulations of 1 minute, and 72 DEG C of final extensions 5 minutes.Use primer cf09024 and cf09025 (for gene 5 ' parts) with cf09026 and cf09027 (for 3 ' parts of gene) from Vector PCR amplification pyr5 gene as isolating mark Thing.PCR reacts usePolymerase (Promega) according to the manufacturer's instructions and uses every kind of primer of 0.2 μM to enter OK.Amplification condition be 95 DEG C 2 minutes, 95 DEG C continue within 30 seconds, 53 DEG C, to continue to continue for 30 seconds, 72 DEG C 35 circulations of 1 minute, and 72 DEG C of final extensions 5 minutes.The primer used shows in table 3-1.In chain overlap extension reaction respectively (see, Horton et al., Meth.Enzymol., 217:270-279 [1993]), will be from primer cf09067 and cf09068 and primer PCR primer that cf09026 and cf09027 obtains merges, to from primer cf09069 and cf09070 and primer cf09024 and The PCR primer that cf09025 obtains is the most such.PCR reaction is by using Finnzymes'sArchaeal dna polymerase according to The explanation of manufacturer is carried out, including 3%DMSO and use every kind of primer of 0.2 μM.Amplification condition be 98 DEG C 1 minute, 98 DEG C continue Within 10 seconds, 62 DEG C, continue 20 seconds, 72 DEG C of 35 circulations continuing 2 minutes, and 72 DEG C of final extensions 5 minutes.Chain overlap extension produces Thing lacks for cdh1.

Embodiment 4

Method for transformation

By C1 cell and derivant bacterial strain with 10⁶The 100ml that spore/ml is inoculated in 500ml Erlenmeyer flask is raw In long culture medium.At 35 DEG C, 250rpm cultivates culture 48 hours.In order to gather in the crops mycelium, culture is filtered through aseptic Myracloth filter (CalBiochem) also uses 100mL1700mosmol NaCl/CaCl₂Solution (0.6M NaCl, 0.27M CaCl₂*H₂O) washing.The mycelium of washing is transferred in 50ml test tube and weighs.By Caylase (20mg/ gram of mycelium; Cayla) 1700mosmol NaCl/CaCl it is dissolved in₂In and ultraviolet sterilization 90sec.Then, by aseptic for 3ml Caylase solution Add in the mycelial test tube comprising washing and mix.Then, by 15ml1700mosmol NaCl/CaCl₂Solution adds examination In pipe and mix.At 30 DEG C, 70rpm cultured mycelia/Caylase suspension 2 hours.By filter through aseptic Myracloth filter Protoplast is gathered in the crops in aseptic 50ml test tube.By cold for 25mL STC (1.2M Sorbitol, 50mM CaCl₂*H₂O,35mM NaCl, 10mM Tris-HCl) add circulation liquid (flow through) and be centrifuged 10min at 4 DEG C with 2720rpm.By little group weight It is suspended in 50mL STC and recentrifuge.After a wash step, little group is resuspended in 1mL STC.

Then, the DNA of each chain overlap extension product of 2 μ g it is pipetted into the bottom of 15mL sterile test tube and adds 1 μ L Aurin tricarboxyli acid (ATA) and 100 μ l protoplast suspension.Mix these contents and protoplast is at room temperature hatched with DNA 25min.Then, 1.7mL PEG4000 solution (60%PEG4000 is added；Polyethylene Glycol, mean molecule quantity 4000 dalton), 50mMCaCl₂·H₂O, 35mM NaCl, 10mM Tris-HCl) and be sufficiently mixed.Solution is kept at room temperature 20 minutes.With STC fills pipe, mixing, and is centrifuged 10min with 2500rpm at 4 DEG C.Pour out STC, and precipitation is resuspended in remaining STC And bed board is on minimum selectivity culture medium flat plate.Flat board is hatched 5 days at 35 DEG C.Bacterium colony is rule again and checks that cdh1 lacks Lose；The bacterium colony with this disappearance is named as bacterial strain " CF-400 ".

Embodiment 5

The confirmation of CDH1 disappearance

As described in Example 3 prepare genomic DNA.By usingPolymerase (Promega) basis The explanation of manufacturer uses every kind of primer (primer cf09112 and cf09113) of 0.2uM to carry out PCR reaction.Amplification condition is 95 DEG C continuing 2 minutes, 95 DEG C continue to continue for 30 seconds, 54 DEG C to continue for 30 seconds, 72 DEG C 35 of 30 seconds circulations and the most downward at 72 DEG C Stretch 5 minutes.Also use primer cf09110 and cf09111 andPolymerase (Promega) makes according to the explanation of manufacturer PCR is carried out with every kind of primer of 0.2uM.Amplification condition be 95 DEG C continue 2 minutes, 95 DEG C continue 30 seconds, 55.4 DEG C continue 30 seconds, 72 DEG C of 35 circulations continuing 30 seconds and final extension 5 minutes at 72 DEG C.These primers are used for single PCR and react with really Recognize and there is not cdh1 gene.Use primer cf09181 and cf09091 to confirm suitable attachment structure and pyr5 mark in PCR The targeting (seeing, table 5-1) of will thing construct.By usingPolymerase (Promega) is according to the explanation of manufacturer The every kind of primer using 0.2uM carries out PCR reaction.Amplification condition is 95 DEG C and continues 2 minutes, and 95 DEG C continue to hold for 30 seconds, 54.4 DEG C Within continuous 30 seconds, 72 DEG C, continue 3 points of 35 circulations of 30 seconds and final extension 5 minutes at 72 DEG C.PCR is produced by agarose gel Thing electrophoresis is to confirm the band pattern of instruction cdh1 disappearance.

Use the disappearance of cdh1 in chlorophenesic acid indole phenol (DCPIP) colorimetric determination test CF-400.By observe with The ability of the reduction DCPIP substrate that parent strain compares minimizing determines the disappearance of cdh1.Cultivate lacking of parent's C1 bacterial strain and presumption The cell losing the bacterial strain of cdh1 the DCPIP testing supernatant activity.In these tests, microtitration plate merges 160 μ The DCPIP reagent solution (being dissolved in 100mM sodium acetate, the 0.2mM DCPIP of pH5.0) of the fresh preparation of L, 20 μ L cellobioses are molten Liquid (being dissolved in the 1g/L cellobiose of deionized water) and the undiluted cell supernatant of 20mL.Measure with kinetics model immediately Passage solution absorbance under 530nm 30 minutes in time, to follow the trail of the loss of the absorbance caused by DCPIP reduction.? SDS-PAGE upper to from demonstrate minimizing reduce DCPIP substrate ability bacterial strain supernatant electrophoresis with confirm do not exist CDH1.The deep-layer liquid using standard operating instructions to be separated from CF-400 and unconverted parent strain by SDS-PAGE is trained The protein of the culture supernatant of hair care ferment.According to the manufacturer's instructions by with Simply Blue Safe Stain (Invitrogen) dyeing observing protein.Unconverted parent strain is observed Cdh1 albumen be～the band of 90kD but It is that CF-400 does not exists.

Embodiment 6

The hydrolysis of corn straw

In these experiments, with ammonium hydroxide aqueous solution, the pH value of the corn straw (NREL) of low-kappa number is adjusted to 5.0.This material is the solid of 41.3%, has the moisture of 58.7%.Beta-dextran content in solid is 40.7%.Acid is pre- The corn straw processed is loaded onto 96 orifice plates and with sodium acetate buffer, is diluted to every hole 110 μ with the 128mM sodium acetate under pH5 The average external volume of L.Total solid load in all experiments is 24.7%, and the concentration of glucosan is 100g glucosan/kg reaction Thing.The enzyme supernatant of CF-200 and CF-400 is used with 3g cellulase/kg reactant.

Due to the existence of glucose free in substrate, in 96 orifice plates, also run one group wherein use water to replace enzyme as right According to hole.The level of this comparison is deducted from the final concentration of glucose measured.Just will once add all of reactive component Plate seal, and place it in the agitator of 55 DEG C and to rotate 73 hours under 950rpm.At the end of reaction, it is allowed to flat board Cooling.Take out sample, dilution, and measure test kit (Sigma) analysis with the generation determining glucose by GO subsequently.In Fig. 2 Provide result.As indicated, CF-200 supernatant produces the glucose of 52.1g/L, and CF-400 supernatant produces 69.4g/L glucose.CF-400 supernatant shows higher saccharifying performance, shows that the disappearance of cdh1 gene decreases saccharifying The gluconic acid formed by glucose during reaction.

Although having illustrated and described the particular of the present invention, but will be significantly for those skilled in the art It is can to carry out multiple other and change and revise without departing from the spirit and scope of the present invention.It is therefore contemplated that the present invention by all this A little changes and amendment are included within the scope of the invention.

The most extensively and briefly describe the present invention.Fall at this total disclosure (the generic Disclosure) individuality (species) and subordinate's classification (subgeneric) packet that each in is narrower also form this A bright part.Can be aptly at any one element being not specifically disclosed the most herein or multiple element, any one limit value Or put into practice invention described herein in the presence of multiple limit value.The most adopted term and statement be used as descriptive term rather than Restrictive.It is intended to the use of these terms and statement and is not excluded for any etc. of described and/or feature that is that show or its part Valency thing, it should be appreciated that in the range of invention required for protection, multiple amendment is possible.Although it will be understood, therefore, that Specifically disclose the present invention by some preferred embodiments and optional feature, but those skilled in the art can profit By the modifications and changes to concept disclosed herein, and these modifications and changes are contemplated as falling within the scope of the present invention.

Claims

1. a fungal cell, described fungal cell is fine to reduce the endogenous secreted by described fungal cell by genetic modification The amount of dimension disaccharide dehydrogenase activity, wherein said fungal cell is bacterial strain CF-400, and wherein said fungal cell comprises fiber two The disappearance of glucocorticoid dehydrogenase 1cdh1 gene and wherein said bacterial strain CF-400 are to modify thermophilic fungus destroyed wire by disappearance cdh1 The derivative strain UV18#100f Δ alp1 Δ pyr5 of (Myceliophthora thermophila) C1 bacterial strain and obtain.

2. fungal cell as claimed in claim 1, wherein said fungal cell is described endogenous for destroying by genetic modification The secreting signal peptide of property cellobiose dehydrogenase.

3. the fungal cell as according to any one of claim 1-2, wherein said fungal cell by genetic modification with reduce The amount of the endogenous cellobiose dehydrogenase expressed by described fungal cell.

4. fungal cell as claimed in claim 3, wherein said fungal cell is described for destroying coding by genetic modification Translation initiation sequence in the transcripton of endogenous cellobiose dehydrogenase.

5. fungal cell as claimed in claim 4, wherein said fungal cell by genetic modification for frameshift mutation is introduced Arrive in the transcripton of coding described endogenous cellobiose dehydrogenase.

6. the fungal cell as according to any one of claim 1-2 and 4-5, wherein said fungal cell by genetic modification with Reduce the transcriptional level of the gene encoding described endogenous cellobiose dehydrogenase.

7. fungal cell as claimed in claim 6, wherein said fungal cell is described for destroying coding by genetic modification The promoter of the gene of endogenous cellobiose dehydrogenase.

8. the fungal cell as according to any one of claim 1-2,4-5 and 7, wherein said fungal cell is by genetic modification For having lacked the gene of coding described endogenous cellobiose dehydrogenase at least in part.

9. the fungal cell as according to any one of claim 1-2,4-5 and 7, wherein said fungal cell is by genetic modification To reduce the catalytic effect of described endogenous cellobiose dehydrogenase.

10. fungal cell as claimed in claim 9, the described endogenous cellobiose dehydrogenase of wherein said fungal cell One or more residues in avtive spot are imitated with the catalysis reducing described endogenous cellobiose dehydrogenase by genetic modification Power.

11. fungal cells as claimed in claim 9, the described endogenous cellobiose dehydrogenase of wherein said fungal cell One or more residues in haemachrome binding structural domain by genetic modification to reduce described endogenous cellobiose dehydrogenase Catalytic effect.

12. fungal cells as claimed in claim 9, wherein said endogenous cellobiose dehydrogenase comprises and SEQ ID NO: The aminoacid sequence that 2 at least 85%, 88%, 90%, 93%, 95%, 97%, 98% or 99% are identical.

13. 1 kinds of enzymatic mixtures, comprise the water-disintegrable enzyme of two or more celluloses, two or more celluloses wherein said Water-disintegrable enzyme is expressed by the fungal cell according to any one of claim 9-12, and described enzymatic mixture comprises and has minimizing The cellobiose dehydrogenase of catalytic effect.

14. enzymatic mixtures as claimed in claim 13, wherein said enzymatic mixture is cell-free mixture.

15. enzymatic mixtures as according to any one of claim 13-14, the substrate of wherein said enzymatic mixture comprises pretreatment Lignocellulose.

16. enzymatic mixtures as claimed in claim 15, the lignocellulose of wherein said pretreatment comprises by pre-selected from acid The lignocellulose of the disposal methods of process, ammonia pretreatment, steam explosion and organic solvent extraction.

17. 1 kinds for the method that produces glucose, including making at least one cellulose matrix with to comprise two or more fine Tieing up the enzymatic mixture contact of plain water-disintegrable enzyme, the wherein said water-disintegrable enzyme of two or more celluloses is by claim 9-12 Fungal cell described in any one expresses, and described enzymatic mixture comprises the cellobiose dehydrogenation of the catalytic effect with minimizing Enzyme.

18. 1 kinds are used for the method producing glucose, arbitrary with claim 13-16 including making at least one cellulose matrix Enzymatic mixture contact described in Xiang.

19. methods as according to any one of claim 17-18, wherein said enzymatic mixture is cell-free mixture.

20. methods as according to any one of claim 17-18, wherein said cellulose matrix is the wood fibre of pretreatment Element.

21. methods as claimed in claim 20, the lignocellulose of wherein said pretreatment comprise by selected from low-kappa number, The lignocellulose of the disposal methods of ammonia pretreatment, steam explosion and organic solvent extraction.

22. methods as according to any one of claim 17-18, also include described glucose fermentation is become end-product.

23. methods as claimed in claim 22, wherein said end-product is fuel alcohol or precursor industrial chemical.

24. methods as claimed in claim 23, wherein said fuel alcohol is ethanol or butanol.

25. fungal cells as according to any one of claim 1-2,4-5,7 and 10-12, also comprise coding to described fungus Cell is the gene of the cellulose degrading enzyme of homology or allos.

26. enzymatic mixtures as according to any one of claim 13-14 and 16, also comprise to described fungal cell for homology or The cellulose degrading enzyme of allos.

27. as claim 17-18,21 and 23-24 according to any one of method, wherein said enzymatic mixture also comprises institute State fungal cell for homology or the cellulose degrading enzyme of allos.

28. 1 kinds of fermentation medium, comprise the fungal cell according to any one of claim 1-12 or 25-27.

29. 1 kinds of fermentation medium, comprise the enzymatic mixture according to any one of claim 13-16.

30. 1 kinds of fermentation medium, comprise the fungal cell according to any one of claim 1-12 or 25-27 and/or right Require the enzymatic mixture according to any one of 13-16.