WO2010060056A2 - Yeast expressing cellulases for simultaneous saccharification and fermentation using cellulose - Google Patents
Yeast expressing cellulases for simultaneous saccharification and fermentation using cellulose Download PDFInfo
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- WO2010060056A2 WO2010060056A2 PCT/US2009/065571 US2009065571W WO2010060056A2 WO 2010060056 A2 WO2010060056 A2 WO 2010060056A2 US 2009065571 W US2009065571 W US 2009065571W WO 2010060056 A2 WO2010060056 A2 WO 2010060056A2
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- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/14—Multiple stages of fermentation; Multiple types of microorganisms or re-use of microorganisms
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- Lignocellulosic biomass is widely recognized as a promising source of raw material for production of renewable fuels and chemicals.
- the primary obstacle impeding the more widespread production of energy from biomass feedstocks is the general absence of low-cost technology for overcoming the recalcitrance of these materials to conversion into useful fuels.
- Lignocellulosic biomass contains carbohydrate fractions (e.g., cellulose and hemicellulose) that can be converted into ethanol. In order to convert these fractions, the cellulose and hemicellulose must ultimately be converted or hydrolyzed into monosaccharides; it is the hydrolysis that has historically proven to be problematic.
- Biomass processing schemes involving enzymatic or microbial hydrolysis commonly involve four biologically mediated transformations: (1) the production of saccharolytic enzymes (cellulases and hemicellulases); (2) the hydrolysis of carbohydrate components present in pretreated biomass to sugars; (3) the fermentation of hexose sugars (e.g., glucose, mannose, and galactose); and (4) the fermentation of pentose sugars (e.g., xylose and arabinose).
- saccharolytic enzymes cellulases and hemicellulases
- carbohydrate components present in pretreated biomass to sugars
- hexose sugars e.g., glucose, mannose, and galactose
- pentose sugars e.g., xylose and arabinose
- CBP consolidated bioprocessing
- CBP offers the potential for lower cost and higher efficiency than processes featuring dedicated cellulase production.
- the benefits result in part from avoided capital costs, substrate and other raw materials, and utilities associated with cellulase production.
- several factors support the realization of higher rates of hydrolysis, and hence reduced reactor volume and capital investment using CBP, including enzyme-microbe synergy and the use of thermophilic organisms and/or complexed cellulase systems.
- cellulose-adherent cellulolytic microorganisms are likely to compete successfully for products of cellulose hydrolysis with non-adhered microbes, e.g., contaminants, which could increase the stability of industrial processes based on microbial cellulose utilization.
- the first type are endoglucanases (1,4- ⁇ -D-glucan 4-glucanohydrolases; EC 3.2.1.4). Endoglucanases cut at random in the cellulose polysaccharide chain of amorphous cellulose, generating oligosaccharides of varying lengths and consequently new chain ends.
- the second type are exoglucanases, including cellodextrinases (1,4- ⁇ -D- glucan glucanohydrolases; EC 3.2.1.74) and cellobiohydrolases (1,4- ⁇ -D-glucan cellobiohydrolases; EC 3.2.1.91).
- Exoglucanases act in a processive manner on the reducing or non-reducing ends of cellulose polysaccharide chains, liberating either glucose (glucanohydrolases) or cellobiose (cellobiohydrolase) as major products. Exoglucanases can also act on microcrystalline cellulose, presumably peeling cellulose chains from the microcrystalline structure.
- the third type are ⁇ -glucosidases ( ⁇ -glucoside glucohydrolases; EC 3.2.1.21).
- ⁇ -Glucosidases hydro lyze soluble cellodextrins and cellobiose to glucose units.
- S. cerevisiae One major shortcoming of S. cerevisiae is its inability to utilize complex polysaccharides such as cellulose, or its break-down products, such as cellobiose and cellodextrins.
- complex polysaccharides such as cellulose
- cellobiose and cellodextrins include cellobiose and cellodextrins.
- heterologous cellulases from bacterial and fungal sources have been transferred to S. cerevisiae, enabling the degradation of cellulosic derivatives (Van Rensburg, P., et al., Yeast 14, 61-16 (1998)), or growth on cellobiose (Van Rooyen, R., et al, J. Biotech. 120, 284-295 (2005)); McBride, J.E., et al., Enzyme Microb. Techol.
- S. cerevisiae Another major shortcoming of the use of S. cerevisiae is that externally produced cellulases function optimally at a higher temperature than the temperature at which S. cerevisiae function optimally. Thus, either the processing must be carried out in a two step process at two different temperatures or one temperature can be selected where both processes function to some extent, but at least one of the processes does not occur at optimal efficiency.
- the present invention provides for heterologous expression of wild-type and codon-optimized combinations of heterologous cellulases in yeast that allows efficient production of ethanol from cellulose sources.
- the invention also provides for expression of such heterologous cellulases in theromtolerant yeast and methods of using such transformed yeast for ethanol production.
- the present invention is directed to cellulytic host cells.
- the host cells of the invention expressing heterologous cellulases and are able to produce ethanol from cellulose.
- the invention provides a transformed thermotolerant yeast host cell comprising at least one heterologous polynucleotide comprising a nucleic acid encoding a cellulase, wherein the yeast host cell is capable of producing ethanol when grown using cellulose as a carbon source.
- the invention provides a transformed thermotolerant yeast host cell comprising: (a) at least one heterologous polynucleotide comprising a nucleic acid which encodes an endoglucanase; (b) at least one heterologous polynucleotide comprising a nucleic acid which encodes a ⁇ -glucosidase; (c) at least one heterologous polynucleotide comprising a nucleic acid which encodes a first cellobiohydrolase; and (d) at least one heterologous polynucleotide comprising a nucleic acid which encodes a second cellobiohydrolase.
- the invention provides a transformed yeast host cell comprising: (a) at least one heterologous polynucleotide comprising a nucleic acid which encodes a cellulase which is an endoglucanase; (b) at least one heterologous polynucleotide comprising a nucleic acid which encodes a cellulase which is a ⁇ - glucosidase; (c) at least one heterologous polynucleotide comprising a nucleic acid which encodes a cellulase which is a first cellobiohydrolase; and (d) at least one heterologous polynucleotide comprising a nucleic acid which encodes a cellulase which is a second cellobiohydrolase, wherein at least two of the cellulases are secreted by the cell.
- the invention provides a transformed yeast host cell comprising at least six heterologous polynucleotides, wherein each heterologous polynucleotide comprises a nucleic acid which encodes a cellulase.
- the invention provides a transformed yeast host cell comprising at least four heterologous polynucleotides, wherein each heterologous polynucleotide comprises a nucleic acid which encodes an endogluconase.
- the invention provides a co-culture comprising at least two yeast host cells wherein (a) at least one of the host cells comprises a first heterologous polynucleotide comprising a nucleic acid which encodes a cellulase which is an endoglucanase; (b) at least one of the host cells comprises a second heterologous polynucleotide comprising a nucleic acid which encodes a cellulase which is a ⁇ - glucosidase; (c) at least one of the host cells comprises a third heterologous polynucleotide comprising a nucleic acid which encodes a cellulase which is a first cellobiohydrolase; (d) at least one of the host cells comprises a fourth heterologous polynucleotide comprising a nucleic acid which encodes a cellulase which is a second cellobiohydrolase; wherein the first polynucleot
- the host cells of the invention comprise a heterologous polynucleotide comprising a nucleic acid encoding a first cellobiohydrolase, a polynucleotide comprising a nucleic acid encoding an endoglucanase, a polynucleotide comprising a nucleic acid encoding a ⁇ -glucosidase and/or a polynucleotide comprising a nucleic acid encoding a second cellobiohydrolase.
- the cellulase, endoglucanase, ⁇ -glucosidase or cellobiohydrolase is a H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Thermobfida fusca, Clostridum thermocellum,
- Clostridium cellulolyticum Clostridum josui, Bacillus pumilis, Cellulomonas fimi,
- Saccharophagus degradans Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinomyces sp., Irpex lacteus,
- thermophilum Acremonium thermophilum, Neosartorya fischeri, Chaetomium globosum, Chaetomium thermophilum, Aspergillus fumigatus, Aspergillus terreus, Neurospora Crassa, or
- Arabidopsis thaliana cellulase, endoglucanase, ⁇ -glucosidase or cellobiohydrolase Arabidopsis thaliana cellulase, endoglucanase, ⁇ -glucosidase or cellobiohydrolase.
- the cellobiohydrolase is an H. grisea CBHl, a T. aurantiacus CBHl, a T. emersonii CBHl, a T. reesei CBHl, a T. emersonii CBH2, a C. lucknowense CBH2 or a T. reesei CBH2.
- the heterologous polynucleotide comprising a nucleic acid which encodes a cellobiohydrolase encodes a fusion protein comprising a cellobiohydrolase and a cellulose binding module (CBM).
- the CBM is the CBM of T.
- the CBM is fused to the cellobiohydrolase via a linker sequence
- the host cell expresses a first and a second cellobiohydrolase, wherein the first cellobiohydrolase is a T. emersonii CBHl and CBD fusion, and the second cellobiohydrolase is a C. lucknowense CBH2b.
- the ⁇ -glucosidase is a S. fibuligera ⁇ - glucosidase.
- the endoglucanase is a C. formosanus endoglucanase.
- the endoglucanse is a T. reesei endoglucanase, e.g. T. reesei EG2.
- At least one or at least two of the cellulases is tethered, In other embodiments of the invention, at least one of the cellulases is secreted, In another embodiment, at least one of the cellulases is tethered and at least one of the cellulases is secreted, In another embodiment, all of the cellulases are secreted.
- the nucleic acid encoding a cellulase is codon optimized.
- the host cell can be a thermotolerant host cell
- the host cell is a Issatchenkia orientalis, Pichia mississippiensis, Pichia mexicana, Pichia farinosa, Clavispora opuntiae, Clavispora lusitaniae, Candida mexicana, Hansenula polymorpha or Kluveryomyces host cell.
- the host cell is a K. lactis or K. marxianus host cell
- the thermotolerant host cell is an S. cerevisiae host cell, wherein the S. cerevisiae is selected to be thermotolerant.
- the host cell can be an oleaginous yeast cell.
- the oleaginous yeast cell is a Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomces, Pythium, Rhodosporidium, Rhodotorula, Trichosporon or Yarrowia cell.
- the host cell is a Saccharomyces cerevisiae cell.
- the host cell can produce ethanol from cellulose at temperatures above about 30° C, 35 °C, 37° C, 42° C, 45° C or 50° C.
- the host cell can produce ethanol at a rate of at least about 10 mg per hour per liter, at least about 30 mg per hour per liter, at least about 40 mg per hour per liter, at least about 50 mg per hour per liter, at least about 60 mg per hour per liter, at least about 70 mg per hour per liter, at least about 80 mg per hour per liter, at least about 90 mg per hour per liter, at least about 100 mg per hour per liter, at least about 200 mg per hour per liter, at least about 300 mg per hour per liter, at least about 400 mg per hour per liter, at least about 500 mg per hour per liter, at least about 600 mg per hour per liter, at least about 700 mg per hour per liter, at least about 800 mg per hour per liter, at least about 900 mg per hour per liter, or at least about 1 g per hour per liter.
- the present invention also provides methods of using the host cells and co- cultures of the invention.
- the present invention is also directed to a method for hydrolyzing a cellulosic substrate, comprising contacting said cellulosic substrate with a host cell or co-culture of the invention.
- the invention is also directed to a method of fermenting cellulose comprising culturing a host cell or co-culture of the invention in medium that contains insoluble cellulose under suitable conditions for a period sufficient to allow saccharification and fermentation of the cellulose.
- the methods further comprise contacting the cellulosic substrate with externally produced cellulase enzymes.
- the cellulosic substrate is a lignocellulosic biomass selected from the group consisting of grass, switch grass, cord grass, rye grass, reed canary grass, miscanthus, sugar-processing residues, sugarcane bagasse, agricultural wastes, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, soybean stover, corn stover, forestry wastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood, softwood, Agave, and combinations thereof.
- lignocellulosic biomass selected from the group consisting of grass, switch grass, cord grass, rye grass, reed canary grass, miscanthus, sugar-processing residues, sugarcane bagasse, agricultural wastes, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat
- the host cell or co-culture produces ethanol.
- the ethanol can be produced at a rate of at least about 10 mg per hour per liter, at least about 30 mg per hour per liter, at least about 40 mg per hour per liter, at least about 50 mg per hour per liter, at least about 60 mg per hour per liter, at least about 70 mg per hour per liter, at least about 80 mg per hour per liter, at least about 90 mg per hour per liter, at least about 100 mg per hour per liter, at least about 200 mg per hour per liter, at least about 300 mg per hour per liter, at least about 400 mg per hour per liter, at least about 500 mg per hour per liter, at least about 600 mg per hour per liter, at least about 700 mg per hour per liter, at least about 800 mg per hour per liter, at least about 900 mg per hour per liter, or at least about 1 g per hour per liter.
- the host cell or co-cultures contact a cellulosic substance at a temperature of at least about 37 °C, least about 42 °C, from about 42 °C to about 45 °C, or from about 42 °C to about 50 °C.
- Figure 1 shows an image of a CMC plate assay to detect endoglucanase I activity in K. lactis (colonies numbered 1-8) and K. marxianus strains (colonies numbered 9-16) transformed with heterologous cellulases. Strains 8 and 16 are untransformed negative controls. The plate on the left shows colony growth, and the plate on the right shows CMCase activity, indicated by the presence of a clearance zone. Clearance zones appear as white spots in the image.
- Figure 2 depicts the results of an MU-lac assay to detect CBHl activity in K. marxianus strains transformed with heterologous cellulases.
- Figure 3 depicts the percent of Avicel converted by several strains of K. marxianus expressing heterologous cellulases.
- Figure 4 depicts the ethanol production/consumption from Avicel by several strains of K. marxianus expressing heterologous cellulases.
- Figure 5 depicts the growth of S. cerevisiae expressing heterologous cellulases on bacterial microcrystalline cellulose (BMCC).
- BMCC bacterial microcrystalline cellulose
- Figure 6 depicts the ethanol production from Avicel by an S. cerevisiae strain expressing heterologous cellulases.
- Figure 7 depicts the ethanol production from pretreated hardwood (5% based on a dry weight percentage) by an S. cerevisiae strain expressing heterologous cellulases.
- Figure 8 depicts the ethanol production from pretreated hardwood (5% based on a dry weight percentage) by an S. cerevisiae expressing heterologous cellulases in the presence of various concentrations of exogenously added cellulases.
- Figure 9 depicts the ethanol production from Avicel by MO288 (circles) and a control strain (triangles) in both YP media and YNB media.
- Figure 10 depicts the ethanol yield from Avicel (15% based on a dry weight percentage) by a small scale simultaneous saccharification and fermentation (SSF) process using S. cerevisiae supplemented with external cellulases.
- the yield from a yeast strain expressing heterologous cellulases (MO288) is compared to the yield from a control strain (MO249) at a variety of external cellulase concentrations over 150 hours. (100% cellulase loading indicates 25 mg/g total solids; initial solids concentration was 15%.)
- Figure 11 depicts the theoretical ethanol yield from a simultaneous saccharification and fermentation (SSF) process using S. cerevisiae supplemented with external cellulases. The yield from a yeast strain expressing heterologous cellulases
- FIG. 12 illustrates the predicted cellulase enzyme savings based on ethanol yield at 168 hours of simultaneous saccharification and fermentation (SSF) process.
- Figure 13 shows the activity of an artificial cellulase in the Avicel conversion assay as described in Example 9. The MO429 strain was transformed the CBHl consensus sequence "CBHlcons," and the MO419 strain was transformed with empty pMU451 vector as a negative control. Descriptions of other strains are found in Table 8 of Example 9. [0045] Figure 14 demonstrates the activity of yeast expressing various combinations of
- Figure 15 demonstrates the activity of yeast expressing various cellulase enzymes on Avicel as described in Example 10.
- Figure 16 depicts the ethanol production from Avicel by a co-culture of five S. cerevisiae strains expressing heterologous cellulases.
- Figure 17 depicts the ethanol production from Avicel by a co-culture of four S. cerevisiae strains expressing heterologous cellulases as well as the ethanol production from strain MO288, which is expressing four cellulases.
- Figure 18 depicts the ethanol production from Avicel by a co-culture of four S.
- Figure 19 depicts the calculated enzyme savings using a co-culture of four S. cerevisiae strains expressing heterologous cellulases or MO288 as compared to untransformed S. cerevisiae.
- Figure 20 depicts the xylose utilization and ethanol production of M0509 freezer stock, YPX-isolate and YPD-isolate.
- Figure 21 depicts the growth of Ml 105 (labeled "colony C2") and MO1046 in the presence of the same medium and 8 g/L acetate at 40 °C.
- Figure 22 depicts the ethanol production by Ml 105 (triangles) and M1088
- Figure 23 depicts the ethanol production of M 1105 where the fermentation was only inoculated with 0.15 g/L DCW and resulted in some sugar accumulation and 29 g/L ethanol.
- Figure 24 depicts the ethanol production of M 1254 is standard IFM (circles) and low ammonium EFM (squares) conditions.
- Figure 25 depicts the specific growth rate of single colonies compared to M1254 and Ml 339 on complex xylose medium supplemented with a synthetic inhibitor mixture (which included 8 g/L acetate) at 40° C. The single colonies were screened at the same conditions as the evolution occurred. Colony Cl was renamed Ml 360.
- Figure 26 depicts the fermentation performance of M 1360 at 40° C on industrially relevant fermentation medium supplemented with glucose. The fermentation was inoculated with 60 mg/L dry cell weight of M1360.
- Figure 27 depicts the ethanol production in SSF runs on PHW (18% solids, unwashed MS 149) at 35° C and 40° C by several strains. All reactions were loaded with 4mg/g "zoomerase" (Novozyme 22c).
- Figure 28 depicts cultures spotted on SC -111 ⁇ plates containing 0.2% of either
- CMC or lichenin or barley- ⁇ -glucan The top two rows of each plate were Y294 based cultures, and the bottom two rows contained MO749 based strains. Numbers indicate the plasmid contained by each strain.
- pMU471 contains the Cf EG and served as positive control. Plates were incubated for 24 hours at 30° C (pictured on the left), after which colonies were washed of and the plates were stained with 0.1% congo red and destained with 1% NaCl (pictured on the right).
- Figure 29 depicts SDS-PAGE analysis of the supernatants of Cel5 cellulase producing strains.
- a strain containing a plasmid with no foreign gene was used as reference strain (REF).
- the strain containing the plasmid pMU471 expressing Cf ⁇ G, the most successful EG previously found was also included.
- Figure 30 depicts the activity of strains expressing EGs on (A) PASC (2 hours) and (B) avicel (24 hours).
- a strain containing a plasmid with no foreign gene was used as reference strain (REF) and the strain expressing C./EG (pMU471) was included as positive control.
- Figure 31 depicts the distribution of avicel conversion ability of yeast supernatants from transformation with 7>EG2 and additional TeCBH lw/7VCBD.
- Ml 088 conversion is presented as a dark vertical line, and the dotted lines flanking this line represent the standard deviation of the measurement.
- Figure 32 depicts the conversion of Avicel in the HTP avicel assay (48 hour time point) by supernatants of cellulase expressing yeast strains.
- M0509 is the negative control expressing no cellulases.
- Strain 1088 is the parental strain expressing only CBHl, CBH2, and BGL, whereas 1179, 1180, and 1181 are transformants of 1088 also expressing 7VEG2.
- Figure 33 depicts ethanol production in paper sludge CBP/SSF with cellulolytic strain M1403 and non-cellulolytic background strain M1254 with various amounts of commercial enzyme supplementation.
- Figure 34 depicts fermentation of two types of paper sludge by CBP yeast
- Figure 35 depicts the performance of cellulolytic yeast strain M0963 and non- cellulolytic control strain (M0509) on 22% unwashed solids of pretreated hardwood (PHW) (MS 149) at various external cellulase concentrations.
- Experimental conditions 22% solids fed batch, pH 5.4, temperature 35°C, all enzyme protein (EP) was "zoomerase” (Novozymes 22C).
- Figure 36 depicts the performance of cellulolytic yeast strain Ml 284 on 30% solids of washed pretreated hardwood at various initial cell loadings.
- Zoomerase Novozymes 22C cellulase preparation
- BGL AB Enzymes EL2008044L BGL preparation
- XyI AB Enzymes EL2007020L xylanase preparation
- Pectinase Genencor Multifect pectinase FE.
- Figure 37 depicts the ethanol production in washed corn stover CBP/SSF with cellulolytic strain M1284 and non-cellulolytic background strain M0509 with various amounts of commercial enzyme supplementation.
- Experimental conditions 18% solids fed batch, lOg/1 cell inoculuation, pH 5.0 and temperature 35°C, 1 mg/g BGL and 1 mg/g xylanase loaded in each case.
- BGL AB Enzymes EL2008044L BGL preparation
- XyI AB Enzymes EL2007020L xylanase preparation.
- Figure 38 depicts the activity on Avicel (A, B) or MULac (C, D) of yeast culture supernatants expressing different CBHl genes, and estimated CBHl concentration (mg/L, E, F) based on MULac.
- the host strain was either Y294 or M0749.
- the CBHl genes are: Te, Talaromyces emersonii; Ct, Chaetomium thermophilum; At, Acremonium thermophilum; Tr, Trichoderma reesei; Hg, Humicola grisea; Ta, Thermoascus aurantiacus.
- the plasmid names are indicated.
- Yeast were cultivated in YPD in triplicate for 3 days. The data are means ⁇ standard deviation.
- Figure 39 shows the genes modified in yeast strain M0509.
- Figure 40 shows the yeast strains used to contract MO5O9 and the relevant genetic modifications.
- Figure 41 shows the genealogy of yeast strain M 1105.
- Figure 42 shows the genealogy of yeast strain M1254.
- a "vector,” e.g., a "plasmid” or “YAC” (yeast artificial chromosome) refers to an extrachromosomal element often carrying one or more genes that are not part of the central metabolism of the cell, and is usually in the form of a circular double-stranded DNA molecule.
- Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.
- the plasmids or vectors of the present invention are stable and self-replicating.
- An "expression vector” is a vector that is capable of directing the expression of genes to which it is operably associated.
- heterologous refers to an element of a vector, plasmid or host cell that is derived from a source other than the endogenous source.
- a heterologous sequence could be a sequence that is derived from a different gene or plasmid from the same host, from a different strain of host cell, or from an organism of a different taxonomic group ⁇ e.g., different kingdom, phylum, class, order, family genus, or species, or any subgroup within one of these classifications).
- the term “heterologous” is also used synonymously herein with the term “exogenous.”
- domain refers to a part of a molecule or structure that shares common physical or chemical features, for example hydrophobic, polar, globular, helical domains or properties, e.g., a DNA binding domain or an ATP binding domain. Domains can be identified by their homology to conserved structural or functional motifs. Examples of cellobiohydrolase (CBH) domains include the catalytic domain (CD) and the cellulose binding domain (CBD).
- CBH cellobiohydrolase
- nucleic acid is a polymeric compound comprised of covalently linked subunits called nucleotides.
- Nucleic acid includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded.
- DNA includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic DNA.
- isolated nucleic acid molecule refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
- nucleic acid molecule refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.
- this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes.
- sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
- a “gene” refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids. “Gene” also refers to a nucleic acid fragment that expresses a specific protein, including intervening sequences (introns) between individual coding segments (exons), as well as regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences.
- a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength.
- Hybridization and washing conditions are well known and exemplified, e.g., in Sambrook, J., Fritsch, E. F. and Maniatis, T.
- One set of conditions uses a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDS at 45°C for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at 50°C for 30 min.
- washes are performed at higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2X SSC, 0.5% SDS are increased to 6O°C.
- Another set of highly stringent conditions uses two final washes in 0.1X SSC, 0.1% SDS at 65°C.
- An additional set of highly stringent conditions are defined by hybridization at 0.1X SSC, 0.1% SDS, 65°C and washed with 2X SSC, 0.1% SDS followed by 0.1X SSC, 0.1% SDS.
- Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
- the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences.
- the relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
- equations for calculating Tm have been derived ⁇ see, e.g., Maniatis at 9.50-9.51).
- the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity ⁇ see, e.g., Maniatis, at 11.7-11.8).
- the length for a hybridizable nucleic acid is at least about 10 nucleotides.
- a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least 30 nucleotides.
- the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.
- identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences.
- identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
- Suitable nucleic acid sequences or fragments thereof encode polypeptides that are at least about 70% to 75% identical to the amino acid sequences reported herein, at least about 80%, 85%, or 90% identical to the amino acid sequences reported herein, or at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences reported herein.
- Suitable nucleic acid fragments are at least about 70%, 75%, or 80% identical to the nucleic acid sequences reported herein, at least about 80%, 85%, or 90% identical to the nucleic acid sequences reported herein, or at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequences reported herein.
- Suitable nucleic acid fragments not only have the above identities/similarities but typically encode a polypeptide having at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, or at least 250 amino acids.
- a DNA or RNA “coding region” is a DNA or RNA molecule which is transcribed and/or translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences.
- Suitable regulatory regions refer to nucleic acid regions located upstream (5' non-coding sequences), within, or downstream (3 1 non- coding sequences) of a coding region, and which influence the transcription, RNA processing or stability, or translation of the associated coding region. Regulatory regions may include promoters, translation leader sequences, RNA processing site, effector binding site and stem-loop structure.
- a coding region can include, but is not limited to, prokaryotic regions, cDNA from mRNA, genomic DNA molecules, synthetic DNA molecules, or RNA molecules. If the coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding region.
- An "isoform” is a protein that has the same function as another protein but which is encoded by a different gene and may have small differences in its sequence.
- a "paralogue” is a protein encoded by a gene related by duplication within a genome.
- orthologue is gene from a different species that has evolved from a common ancestral gene by speciation. Normally, orthologues retain the same function in the course of evolution as the ancestral gene.
- ORF Open reading frame
- nucleic acid either DNA, cDNA or RNA, that comprises a translation start signal or initiation codon, such as an ATG or AUG, and a termination codon and can be potentially translated into a polypeptide sequence.
- Promoter refers to a DNA fragment capable of controlling the expression of a coding sequence or functional RNA.
- a coding region is located 3' to a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”.
- a promoter is generally bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease Sl), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
- a coding region is "under the control" of transcriptional and translational control elements in a cell when RNA polymerase transcribes the coding region into mRNA, which is then trans-RNA spliced (if the coding region contains introns) and translated into the protein encoded by the coding region.
- Transcriptional and translational control regions are DNA regulatory regions, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding region in a host cell.
- polyadenylation signals are control regions.
- operably associated refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
- a promoter is operably associated with a coding region when it is capable of affecting the expression of that coding region ⁇ i.e., that the coding region is under the transcriptional control of the promoter). Coding regions can be operably associated to regulatory regions in sense or antisense orientation.
- expression refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.
- the present invention provides host cells expressing heterologous cellulases that can be effectively and efficiently utilized to produce ethanol from cellulose.
- the host cells can be a yeast.
- the yeast host cell can be, for example, from the genera Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, and Yarrowia.
- Yeast species as host cells may include, for example, S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K.
- the yeast is selected from the group consisting of Saccharomyces cerevisiae, Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe and Schwanniomyces occ ⁇ dentalis.
- the yeast is Saccharomyces cerevisiae.
- the yeast is a thermotolerant Saccharomyces cerevisiae. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
- the host cell is an oleaginous cell.
- the oleaginous host cell can be an oleaginous yeast cell.
- the oleaginous yeast host cell can be from the genera Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidum, Rhodotorula, Trichosporon or Yarrowia.
- the oleaginous host cell can be an oleaginous microalgae host cell.
- the oleaginous microalgea host cell can be from the genera Thraustochytrium or Schizochytrium.
- Biodiesel could then be produced from the triglyceride produced by the oleaginous organisms using conventional lipid transesterification processes.
- the oleaginous host cells can be induced to secrete synthesized lipids.
- Embodiments using oleaginous host cells are advantegeous because they can produce biodiesel from lignocellulosic feedstocks which, relative to oilseed substrates, are cheaper, can be grown more densely, show lower life cycle carbon dioxide emissions, and can be cultivated on marginal lands.
- the host cell is a thermotolerant host cell.
- Thermotolerant host cells can be particularly useful in simultaneous saccharification and fermentation processes by allowing externally produced cellulases and ethanol-producing host cells to perform optimally in similar temperature ranges.
- Thermotolerant host cells of the invention can include, for example, Issatchenkia orientalis, Pichia mississippiensis, Pichia mexicana, Pichia farinosa, Clavispora opuntiae, Clavispora lusitaniae, Candida mexicana, Hansenula polymorpha and Kluyveromyces host cells.
- the thermotolerant cell is an S. cerevisiae strain, or other yeast strain, that has been adapted to grow in high temperatures, for example, by selection for growth at high temperatures in a cytostat.
- the host cell is a
- the Kluyveromyces host cell can be a K. lactis, K. marxianus, K. blattae, K. phaffii, K. yarrowii, K. aestuarii, K. dobzhanskii, K. wickerhamii K. thermotolerans, or K. waltii host cell.
- the host cell is a K. lactis, or K. marxianus host cell, In another embodiment, the host cell is a K. marxianus host cell.
- thermotolerant host cell can grow at temperatures above about 30° C, about 31° C, about 32° C, about 33° C, about 34° C, about 35° C, about 36° C, about 37° C, about 38° C, about 39° C, about 40° C, about 41° C or about 42° C.
- thermotolerant host cell can produce ethanol from cellulose at temperatures above about 30° C, about 31° C, about 32° C, about 33° C, about 34° C, about 35° C, about 36° C, about 37° C, about 38° C, about 39° C, about 40° C, about 41° C, about 42° C, or about 43 °C, or about 44 °C, or about 45 °C, or about 50° C.
- the thermotolerant host cell can grow at temperatures from about 30° C to 60° C, about 30° C to 55° C, about 30° C to 50° C, about 40° C to 60° C, about 40° C to 55° C or about 40° C to 50° C.
- the thermotolterant host cell can produce ethanol from cellulose at temperatures from about 30° C to 60° C, about 30° C to 55° C, about 30° C to 50° C, about 40° C to 60° C, about 40° C to 55° C or about 40° C to 50° C.
- the host cell has the ability to metabolize xylose.
- xylose-utilizing technology can be found in the following publications: Kuyper M et al. FEMS Yeast Res. 4: 655-64 (2004), Kuyper M et al. FEMS Yeast Res. 5:399-409 (2005), and Kuyper M et al. FEMS Yeast Res. 5:925-34 (2005), which are herein incorporated by reference in their entirety.
- xylose-utilization can be accomplished in S. cerevisiae by heterologously expressing the xylose isomerase gene, XyIA, e.g.
- the host cells can contain antibiotic markers or can contain no antibiotic markers.
- Host cells are genetically engineered (transduced or transformed or transfected) with the polynucleotides encoding cellulases of this invention which are described in more detail below.
- the polynucleotides encoding cellulases can be introduced to the host cell on a vector of the invention, which may be, for example, a cloning vector or an expression vector comprising a sequence encoding a heterologous cellulase.
- the host cells can comprise polynucleotides of the invention as integrated copies or plasmid copies.
- the present invention relates to host cells containing the polynucleotide constructs described below.
- the host cells of the present invention can express one or more heterologous cellulase polypeptides.
- the host cell comprises a combination of polynucleotides that encode heterologous cellulases or fragments, variants or derivatives thereof.
- the host cell can, for example, comprise multiple copies of the same nucleic acid sequence, for example, to increase expression levels, or the host cell can comprise a combination of unique polynucleotides,
- the host cell comprises a single polynucleotide that encodes a heterologous cellulase or a fragment, variant or derivative thereof.
- host cells expressing a single heterologous cellulase can be used in co-culture with other host cells of the invention comprising a polynucleotide that encodes at least one other heterologous cellulase or fragment, variant or derivative thereof.
- Introduction of a polynucleotide encoding a heterologous cellulase into a host cell can be done by methods known in the art.
- Introduction of polynucleotides encoding heterologous cellulases into, for example yeast host cells can be effected by lithium acetate transformation, spheroplast transformation, or transformation by electroporation, as described in Current Protocols in Molecular Biology, 13.7.1-13.7.10.
- Introduction of the construct in other host cells can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation. (Davis, L., et ah, Basic Methods in Molecular Biology, (1986)).
- the transformed host cells or cell cultures, as described above, can be examined for endoglucanase, cellobiohydrolase and/or ⁇ glucosidase protein content.
- protein content can be determined by analyzing the host (e.g., yeast) cell supernatants.
- host e.g., yeast
- high molecular weight material can be recovered from the yeast cell supernatant either by acetone precipitation or by buffering the samples with disposable de-salting cartridges.
- Proteins including tethered heterologous cellulases, can also be recovered and purified from recombinant yeast cell cultures by methods including spheroplast preparation and lysis, cell disruption using glass beads, and cell disruption using liquid nitrogen for example. Additional protein purification methods include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, gel filtration, and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
- HPLC high performance liquid chromatography
- Protein analysis methods include methods such as the traditional Lo wry method or the protein assay method according to BioRad's manufacturer's protocol. Using such methods, the protein content of saccharolytic enzymes can be estimated. Additionally, to accurately measure protein concentration a heterologous cellulase can be expressed with a tag, for example a His-tag or HA-tag and purified by standard methods using, for example, antibodies against the tag, a standard nickel resin purification technique or similar approach.
- a tag for example a His-tag or HA-tag
- purified using, for example, antibodies against the tag, a standard nickel resin purification technique or similar approach.
- the transformed host cells or cell cultures, as described above, can be further analyzed for hydrolysis of cellulose (e.g., by a sugar detection assay), for a particular type of cellulase activity (e.g., by measuring the individual endoglucanase, cellobiohydrolase or ⁇ glucosidase activity) or for total cellulase activity.
- Endoglucanase activity can be determined, for example, by measuring an increase of reducing ends in an endoglucanase specific CMC substrate.
- Cellobiohydrolase activity can be measured, for example, by using insoluble cellulosic substrates such as the amorphous substrate phosphoric acid swollen cellulose (PASC) or microcrystalline cellulose (Avicel) and determining the extent of the substrate's hydrolysis, ⁇ -glucosidase activity can be measured by a variety of assays, e.g., using cellobiose.
- PASC amorphous substrate phosphoric acid swollen cellulose
- Avicel microcrystalline cellulose
- a total cellulase activity which includes the activity of endoglucanase, cellobiohydrolase and ⁇ -glucosidase, can hydrolyze crystalline cellulose synergistically.
- Total cellulase activity can thus be measured using insoluble substrates including pure cellulosic substrates such as Whatman No. 1 filter paper, cotton linter, microcrystalline cellulose, bacterial cellulose, algal cellulose, and cellulose-containing substrates such as dyed cellulose, alpha-cellulose or pretreated lignocellulose.
- cellulases can also be detected by methods known to one of ordinary skill in the art, such as by the Avicel assay (described supra) that would be normalized by protein (cellulase) concentration measured for the sample.
- a cellulase can be any enzyme involved in cellulase digestion, metabolism and/or hydrolysis, including an endoglucanase, exogluconase, or ⁇ -glucosidase.
- the transformed host cells or cell cultures are assayed for ethanol production. Ethanol production can be measured by techniques known to one or ordinary skill in the art e.g. by a standard HPLC refractive index method.
- the expression of heterologous cellulases in a host cell can be used advantageously to produce ethanol from cellulosic sources.
- Cellulases from a variety of sources can be heterologously expressed to successfully increase efficiency of ethanol production.
- the cellulases can be from fungi, bacteria, plant, protozoan or termite sources.
- the cellulase is a H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N.
- multiple cellulases from a single organism are co-expressed in the same host cell.
- multiple cellulases from different organisms are co-expressed in the same host cell.
- cellulases from two, three, four, five, six, seven, eight, nine or more organisms can be co- expressed in the same host cell.
- the invention can encompass co-cultures of yeast strains, wherein the yeast strains express different cellulases.
- Co-cultures can include yeast strains expressing heterologous cellulases from the same organisms or from different organisms.
- Co-cultures can include yeast strains expressing cellulases from two, three, four, five, six, seven, eight, nine or more organisms.
- Cellulases of the present invention include both endoglucanases or exoglucanases.
- the cellulases can be, for example, endoglucanases, ⁇ -glucosidases or cellobiohydrolases.
- the endoglucanase(s) can be an endoglucanase I or an endoglucanase II isoform, paralogue or orthologue.
- the endoglucanase expressed by the host cells of the present invention can be recombinant endo-1,4- ⁇ -glucanase.
- the endoglucanase is a T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, R.
- the endoglucanase comprises an amino acid sequence selected from SEQ ID NOs: 30-39 or 52-56, as shown in Table 1 below, In certain other embodiments, the endoglucanase comprises an amino acid sequence that is at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to an amino acid sequence selected from SEQ ID NOs: 30-39 or 52-56.
- 95%, 96%, 97%, 98%, 99% or 100% identical to a polypeptide of the present invention can be determined conventionally using known computer programs. Methods for determining percent identity, as discussed in more detail below in relation to polynucleotide identity, are also relevant for evaluating polypeptide sequence identity.
- the endoglucanase is an endoglucanase I ("egl") from Trichoderma reesei.
- the endoglucanase comprises an amino acid sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ E) NO:39.
- the endoglucanase is an endoglucanase from C. formosanus.
- the endoglucanase comprises an amino acid sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:31.
- the endoglucanase is an endoglucanase from H. jecorina.
- the endoglucanase comprises an amino acid sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ BD NO:54.
- the ⁇ -glucosidase is a ⁇ -glucosidase I or a ⁇ -glucosidase
- the ⁇ -glucosidase is derived from Saccharomycopsis fibuligera.
- the ⁇ -glucosidase comprises an amino acid sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:40.
- the cellobiohydrolase(s) can be a cellobiohydrolase I and/or a cellobiohydrolase II isoform, paralogue or orthologue.
- the cellobiohydrolase comprises an amino acid sequence selected from SEQ ID NOs: 21-29 or 46, as shown in Table 1 below.
- the cellobiohydrolase is a cellobiohydrolase I or II from Trichoderma reesei.
- the cellobiohydrolase comprises a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:27 or SEQ ID NO:28.
- the cellobiohydrolase is a cellobiohydrolase I or II from T. emersonii.
- the cellobiohydrolase comprises a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:23 or SEQ E) NO:24.
- the cellobiohydrolase of the invention is a C. lucknowense cellobiohydrolase.
- the cellobiohydrolase is C. lucknowense cellobiohydrolase Cbh2b.
- the cellobiohydrolase comprises a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:25.
- the cellulase comprises a sequence selected from the sequences in Table 1 below.
- the cellulases of the invention also include cellulases that comprise a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99 or 100% identical to the sequences of Table 1.
- Some embodiments of the invention encompass a polypeptide comprising at least
- polypeptides and polynucleotides of the present invention are provided in an isolated form, e.g., purified to homogeneity.
- the present invention also encompasses polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% similar to the polypeptide of any of SEQ E) NOs: 21-40, 46, or 52-56 and to portions of such polypeptide with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
- the present invention further relates to a domain, fragment, variant, derivative, or analog of the polypeptide of any of SEQ ID NOs: 21-40, 46, or 52-56.
- fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis. Therefore, the fragments may be employed as intermediates for producing the full-length polypeptides.
- Fragments of cellobiohydrolase, endoglucanase or beta-glucosidase polypeptides encompass domains, proteolytic fragments, deletion fragments and in particular, fragments of H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. ⁇ buligera, C. lucknowense, R.
- Clostridum thermocellum Clostridium cellulolyticum
- Clostridum josui Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H.
- Polypeptide fragments further include any portion of the polypeptide which retains a catalytic activity of cellobiohydrolase, endoglucanase or beta- glucosidase proteins.
- 46, or 52-56 may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide for purification of the polypeptide or (v) one in which a fragment of the polypeptide is soluble, i.e., not membrane bound, yet still binds ligands to the membrane bound receptor.
- a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
- substituted amino acid residue may or may not be one encoded
- the polypeptides of the present invention further include variants of the polypeptides.
- a "variant" of the polypeptide can be a conservative variant, or an allelic variant.
- a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the protein.
- a substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the protein.
- the overall charge, structure or hydrophobic-hydrophilic properties of the protein can be altered without adversely affecting a biological activity.
- the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the protein.
- an "allelic variant” is intended alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques. Allelic variants, though possessing a slightly different amino acid sequence than those recited above, will still have the same or similar biological functions associated with the H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M.
- darwinensis N. walkeri, S. fibuligera, C. lucknowense, R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H.
- allelic variants, the conservative substitution variants, and members of the endoglucanase, cellobiohydrolase or ⁇ -glucosidase protein families can have an amino acid sequence having at least 75%, at least 80%, at least 90%, at least 95% amino acid sequence identity with a H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R.
- the proteins and peptides of the present invention include molecules comprising the amino acid sequence of SEQ ED NOs: 21-40, 46 and 52-56 or fragments thereof having a consecutive sequence of at least about 3, 4, 5, 6, 10, 15, 20, 25, 30, 35 or more amino acid residues of the H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R.
- speratus Aspergillus kawachii, Heterodera schachtii, H. jecorina, Orpinomyces sp., Irpex lacteus, Acremonium thermophilum, R. flavipes, or Neosartorya fischeri cellobiohydrolase, endoglucanase or beta-glucosidase polypeptide sequences; amino acid sequence variants of such sequences wherein at least one amino acid residue has been inserted N- or C- terminal to, or within, the disclosed sequence; amino acid sequence variants of the disclosed sequences, or their fragments as defined above, that have been substituted by another residue.
- Contemplated variants further include those containing predetermined mutations by, e.g., homologous recombination, site-directed or PCR mutagenesis, and the corresponding proteins of other animal species, including but not limited to bacterial, fungal, insect, rabbit, rat, porcine, bovine, ovine, equine and non-human primate species, the alleles or other naturally occurring variants of the family of proteins; and derivatives wherein the protein has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example, a detectable moiety such as an enzyme or radioisotope).
- a detectable moiety such as an enzyme or radioisotope
- variants may be generated to improve or alter the characteristics of the cellulase polypeptides. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function.
- the invention further includes H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R.
- Clostridum thermocellum Clostridium cellulolyticum
- Clostridum josui Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H.
- the second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can then be tested for biological activity.
- tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, VaI, Leu and He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
- derivatives and analogs refer to a polypeptide differing from the H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R.
- Clostridum thermocellum Clostridium cellulolyticum
- Clostridum josui Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H.
- aurantiacus T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. lucknowense, R.
- Clostridum thermocellum Clostridium cellulolyticum
- Clostridum josui Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H.
- Derivatives can be covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example, a detectable moiety such as an enzyme or radioisotope).
- a detectable moiety such as an enzyme or radioisotope.
- Examples of derivatives include fusion proteins.
- An analog is another form of a H. grisea, T. aurantiacus, T. emersonii, T. reesei,
- An "analog” also retains substantially the same biological function or activity as the polypeptide of interest, e.g., functions as a cellobiohydrolase.
- An analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
- the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, In some particular embodiments, the polypeptide is a recombinant polypeptide.
- allelic variants, orthologs, and/or species homologs are also provided in the present invention. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to any of SEQ ID NOs: 1-40, using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
- the host cells express at least one heterologous cellulase that is not derived from any one particular organism, but instead has an artificial amino acid sequence that is a consensus cellulase sequence.
- the consensus cellulase sequence can be an endoglucanase consensus sequence, a ⁇ - glucosidase consensus sequence, or a cellobiohydrolase consensus sequence.
- the heterologous cellulase is a CBHl consensus sequence. Therefore, in one embodiment, the invention is directed to a polypeptide sequence which comprises a sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to the consensus CBHl sequence of SEQ ID NO: 43. In some embodiments, the invention is directed to a polypeptide which comprises the sequence of SEQ ID NO: 43.
- the invention is also directed to host cells that comprise a polypeptide sequence which comprises a sequence that is at least 80%, 85%, 90%, 95%, 98% 99% or 100% identical to the consensus CBHl sequence of SEQ ID NO: 43.
- the invention further directed to host cells that comprise a polynucleotide that encodes a polypeptide sequence which comprises a sequence that is at least 80%, 85%, 90%, 95%, 98% 99% or 100% identical to the consensus CBHl sequence of SEQ ID NO: 43.
- the host cell comprises at least one polynucleotide that encodes a polypeptide sequence which comprises a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the consensus CBHl sequence of SEQ ID NO: 43 and at least a second polynucleotide that encodes a heterologous cellulase.
- the second polynucleotide can encode a endoglucanase, a ⁇ -glucosidase, a cellobiohydrolase, an endoglucanase consensus sequence, a ⁇ -glucosidase consensus sequence, or a cellobiohydrolase consensus sequence
- the host cell comprising the polynucleotide that encodes a polypeptide sequence which comprises a sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the consensus CBHl sequence of SEQ ID NO: 43 is capable of producing ethanol when grown using cellulose as a carbon source.
- the host cells express a combination of heterologous cellulases.
- the host cell can contain at least two heterologous cellulases, at least three heterologous cellulases, at least four heterologous cellulases, at least five heterologous cellulases, at least six heterologous cellulases, at least seven heterologous cellulases, at least eight heterologous cellulases, at least nine heterologous cellulases, at least ten heterologous cellulases, at least eleven heterologous cellulases, at least twelve heterologous cellulases, at least thirteen heterologous cellulases, at least fourteen heterologous cellulases or at least fifteen heterologous cellulases.
- the heterologous cellulases in the host cell can be from the same or from different species.
- the host cells express a combination of heterologous cellulases which includes at least one endoglucanase, at least one ⁇ -glucosidase and at least one cellobiohydrolase.
- the host cells express a combination of heterologous cellulases which includes at least one endoglucanase, at least one ⁇ -glucosidase and at least two cellobiohydrolases.
- the at least two cellobiohydrolases can be both be cellobiohydrolase I, can both be cellobiohydrolase II, or can be one cellobiohydrolase I and one cellobiohydrolase II.
- the host cells express a combination of cellulases that includes a C. formosanus endoglucanase I and an S. fibuligera ⁇ -glucosidase I.
- the host cells express a combination of cellulases that includes a T. emersonii cellobiohydrolase I, and a T. reesei cellobiohydrolase II.
- the host cells express a combination of cellulases that includes a C. formosanus endoglucanase I, an S. fibuligera ⁇ -glucosidase I, a T. emersonii cellobiohydrolase I, and a C. lucknowense cellobiohydrolase lib.
- the host cells express a combination of cellulases that includes a C. formosanus endoglucanase I, an S. fibuligera ⁇ -glucosidase I, a T. emersonii cellobiohydrolase I, and a T. reesei cellobiohydrolase II.
- the host cells express a combination of cellulases that includes an H. jecorina endogluconase 2, an S. fibuligera ⁇ -glucosidase I, a T. emersonii cellobiohydrolase I, and a T. reesei cellobiohydrolase II.
- the host cells express a combination of cellulases that includes an H. jecorina endogluconase 2, an S. fibuligera ⁇ -glucosidase I, a T. emersonii cellobiohydrolase I, and a C. lucknowense cellobiohydrolase II. Tethered and Secreted Cellulases
- the cellulases may be either tethered or secreted.
- a protein is "tethered" to an organism's cell surface if at least one terminus of the protein is bound, covalently and/or electrostatically for example, to the cell membrane or cell wall.
- a tethered protein may include one or more enzymatic regions that may be joined to one or more other types of regions at the nucleic acid and/or protein levels ⁇ e.g., a promoter, a terminator, an anchoring domain, a linker, a signaling region, etc.). While the one or more enzymatic regions may not be directly bound to the cell membrane or cell wall (e.g., such as when binding occurs via an anchoring domain), the protein is nonetheless considered a "tethered enzyme" according to the present specification.
- Tethering may, for example, be accomplished by incorporation of an anchoring domain into a recombinant protein that is heterologously expressed by a cell, or by prenylation, fatty acyl linkage, glycosyl phosphatidyl inositol anchors or other suitable molecular anchors which may anchor the tethered protein to the cell membrane or cell wall of the host cell.
- a tethered protein maybe tethered at its amino terminal end or optionally at its carboxy terminal end.
- secreted means released into the extracellular milieu, for example into the media.
- tethered proteins may have secretion signals as part of their immature amino acid sequence, they are maintained as attached to the cell surface, and do not fall within the scope of secreted proteins as used herein.
- flexible linker sequence refers to an amino acid sequence which links two amino acid sequences, for example, a cell wall anchoring amino acid sequence with an amino acid sequence that contains the desired enzymatic activity.
- the flexible linker sequence allows for necessary freedom for the amino acid sequence that contains the desired enzymatic activity to have reduced steric hindrance with respect to proximity to the cell and may also facilitate proper folding of the amino acid sequence that contains the desired enzymatic activity.
- the tethered cellulase enzymes are tethered by a flexible linker sequence linked to an anchoring domain.
- the anchoring domain is of CWP2 (for carboxy terminal anchoring) or FLOl (for amino terminal anchoring) from S. cerevisiae.
- heterologous secretion signals may be added to the expression vectors of the present invention to facilitate the extra-cellular expression of cellulase proteins.
- the heterologous secretion signal is the secretion signal from T. reesei Xyn2. Fusion Proteins Comprising Cellulases
- the present invention also encompasses fusion proteins.
- the fusion proteins can be a fusion of a heterologous cellulase and a second peptide.
- the heterologous cellulase and the second peptide can be fused directly or indirectly, for example, through a linker sequence.
- the fusion protein can comprise for example, a second peptide that is N-terminal to the heterologous cellulase and/or a second peptide that is C-terminal to the heterologous cellulase.
- the polypeptide of the present invention comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a heterologous cellulase.
- the fusion protein can comprise a first and second polypeptide wherein the first polypeptide comprises a heterologous cellulase and the second polypeptide comprises a signal sequence.
- the fusion protein can comprise a first and second polypeptide, wherein the first polypeptide comprises a heterologous cellulase and the second polypeptide comprises a polypeptide used to facilitate purification or identification or a reporter peptide.
- the polypeptide used to facilitate purification or identification or the reporter peptide can be, for example, a HIS-tag, a GST-tag, an HA-tag, a FLAG-tag, a MYC-tag, or a fluorescent protein.
- the fusion protein can comprise a first and second polypeptide, wherein the first polypeptide comprises a heterologous cellulase and the second polypeptide comprises an anchoring peptide.
- the anchoring domain is of CWP2 (for carboxy terminal anchoring) or FLOl (for amino terminal anchoring) from S. cerevisiae.
- the fusion protein can comprise a first and second polypeptide, wherein the first polypeptide comprises a heterologous cellulase and the second polypeptide comprises a cellulose binding module (CBM).
- CBM is from, for example, T. reesei Cbhl or Cbh2, from H. grisea Cbhl, or from C. lucknowense Cbh2b.
- the CBM is fused to a cellobiohydrolase.
- the fusion protein comprises a first and second polypeptide, wherein the first polypeptide comprises a heterologous cellobiohydrolase and the second polypeptide comprises a CBM.
- the cellobiohydrolase is T. emersonii cellobiohydrolase I and the CBM is a T. reesei cellobiohydrolase CBM.
- the cellobiohydrolase is T. emersonii cellobiohydrolase I and the CBM is a H. grisea cellobiohydrolase CBM.
- the CBM of H. grisea comprises amino acids 492-525 of SEQ ID NO: 21.
- the polypeptide of the present invention encompasses a fusion protein comprising a first polypeptide and a second polypeptide, wherien the first polypeptide is a cellobiohydrolase, and the second polypeptide is a domain or fragment of a cellobiohydrolase.
- the polypeptide of the present invention encompasses a fusion protein comprising a first polypeptide, where the first polypeptide is a T. emersonii Cbhl , H. grisea Cbhl, T. aurantiacusi Cbhl, T. emersonii Cbh2, T. reesei Cbhl T.
- the first polypeptide is T. emersonii Cbhl and the second polynucleotide is a CBM from T.
- the first polypeptide is either N- terminal or C-terminal to the second polypeptide.
- the first polypeptide and/or the second polypeptide are encoded by codon-optimized polynucleotides, for example, polynucleotides codon-optimized for S. cerevisiae or Kluveromyces.
- the first polynucleotide is a codon-optimized T. emersonii cbhl and the second polynucleotide encodes for a codon-optimized CBM from T.
- the first polynucleotide is a codon-optimized T. emersonii cbhl and the second polynucleotide encodes for a codon-optimized CBM from C. lucknowense or Cbh2b.
- the first polypeptide and the second polypeptide are fused via a linker sequence.
- the linker sequence can, in some embodiments, be encoded by a codon-optimized polynucelotide.
- Codon-optimized polynucleotides are described in more detail below.
- An amino acid sequence corresponding to a codon-optimized linker 1 according to the invention is a flexible linker - strep tag - TEV site - FLAG - flexible linker fusion and corresponds to GGGGSGGGGS AW ⁇ PQFGG ENLYFQG DYKDDDK GGGGSGGGGS (SEQ ID NO:57) [0172]
- the DNA sequence is as follows:
- An amino acid sequence corresponding to optimized linker 2 is a flexible linker - strep tag -linker- TEV site - flexible linker and corresponds to GGGGSGGGGS WSHPQFEK GG ENLYFQG GGGGSGGGGS (SEQ ID NO:58).
- the DNA sequence is as follows:
- the present invention is also directed to co-cultures comprising at least two yeast host cells wherein the at least two yeast host cells each comprise an isolated polynucleotide encoding a heterologous cellulase.
- co-culture refers to growing two different strains or species of host cells together in the same vessel.
- At least one host cell of the co-culture comprises a heterologous polynucleotide comprising a nucleic acid which encodes an endoglucanase
- at least one host cell of the co-culture comprises a heterologous polynucleotide comprising a nucleic acid which encodes a ⁇ -glucosidase
- at least one host cell comprises a heterologous polynucleotide comprising a nucleic acid which encodes a cellobiohydrolase.
- the co-culture further comprises a host cell comprising a heterologous polynucleotide comprising a nucleic acid which encodes a second cellobiohydrolase.
- the co-culture can comprise two or more strains of yeast host cells and the heterologous cellulases can be expressed in any combination in the two or more strains of host cells.
- the co-culture can comprise two strains: one strain of host cells that expresses an endoglucanase and a second strain of host cells that expresses a ⁇ -glucosidase, a cellobiohydrolase and a second cellobiohydrolase.
- the co-culture can also comprise four strains: one strain of host cells which expresses an endoglucanase, one strain of host cells that expresses a ⁇ -glucosidase, one strain of host cells which expresses a first cellobiohydrolase, and one strain of host cells which expressess a second cellobiohydrolase.
- the co-culture can comprise one strain of host cells that expresses two cellulases, for example an endoglucanase and a beta-glucosidase and a second strain of host cells that expresses one or more cellulases, for example one or more cellobiohydrolases.
- the co-culture can, in addition to the at least two host cells comprising heterologous cellulases, also include other host cells which do not comprise heterologous cellulases.
- the various host cell strains in the co-culture can be present in equal numbers, or one strain or species of host cell can significantly outnumber another second strain or species of host cells.
- the ratio of one host cell to another can be about 1 :1, 1:2, 1 :3, 1 :4, 1:5, 1:10, 1:100, 1 :500 or 1:1000.
- the strains or species of host cells may be present in equal or unequal numbers.
- the co-cultures of the present invention can include tethered cellulases, secreted cellulases or both tethered and secreted cellulases.
- the co-culture comprises at least one yeast host cell comprising a polynucleotide encoding a secreted heterologous cellulase.
- the co-culture comprises at least one yeast host cell comprising a polynucleotide encoding a tethered heterologous cellulase.
- all of the heterologous cellulases in the co-culture are secreted, and in another embodiment, all of the heterologous cellulases in the co-culture are tethered. In addition, other cellulases, such as externally added cellulases may be present in the co-culture.
- the present invention also includes isolated polynucleotides encoding cellulases of the present invention.
- the polynucleotides of the invention can encode endoglucanases or exoglucanases.
- the polynucleotides can encode endoglucanases, ⁇ - glucosidases or cellobiohydrolases.
- the polynucleotide encodes an endoglucanase which is an endo-1,4- ⁇ -glucanase.
- the polynucleotide encodes an endoglucanase I from Trichoderma reesei.
- the endoglucanase is encoded by a polynucleotide comprising a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO: 19.
- the polynucleotide encodes an endoglucanase I from C. formosanus.
- the endoglucanase is encoded by a polynucleotide comprising a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:11.
- the polynucleotide encodes an endoglucanase I from Trichoderma reesei.
- the endoglucanase is encoded by a polynucleotide comprising a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO: 19.
- the polynucleotide encodes an endoglucanase 2 from H. jecorina.
- the endoglucanase is encoded by a polynucleotide comprising a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:54.
- the polynucleotide encodes a ⁇ -glucosidase I or a ⁇ - glucosidase II isoform, paralogue or orthologue.
- the polynucleotide encodes a ⁇ -glucosidase derived from Saccharomycopsis fibuligera.
- the ⁇ -glucosidase is encoded by a polynucleotide comprising a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:20.
- the polynucleotide encodes a cellobiohydrolase I and/or an cellobiohydrolase II isoform, paralogue or orthologue.
- the polynucleotide encodes the cellobiohydrolase I or II from Trichoderma reesei.
- the polynucleotide encodes the cellobiohydrolase I or II from Trichoderma emersonii.
- the cellobiohydrolase is encoded by a polynucleotide comprising a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:7 or SEQ ID NO:8.
- the polynucleotide encodes a cellobiohydrolase from C. lucknowense.
- the cellobiohydrolase is encoded by a polynucleotide comprising a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to SEQ ID NO:5.
- the polynucleotide is a polypeptide comprising a sequence at least about 70, about 80, about 90, about 95, about 96, about 97, about 98, about 99, or 100% identical to a nucleotide sequence listed in Table 1.
- the polynucleotide can encode an endoglucanase, cellobiohydrolase or ⁇ -glucosidase derived from, for example, a fungal, bacterial, protozoan or termite source.
- the present invention relates to a polynucleotide comprising a nucleic acid encoding a functional or structural domain of T. emersonii, H. grisea, T. aurantiacus, C. lucknowense or T. reesei Cbhl or Cbh2.
- T. emersonii H. grisea
- T. aurantiacus C. lucknowense
- T. reesei Cbhl or Cbh2 the domains of T.
- reesei Cbh 1 include, without limitation: (1) a signal sequence, from amino acid 1 to 33 of SEQ ID NO: 27; (2) a catalytic domain (CD) from about amino acid 41 to about amino acid 465 of SEQ ID NO: 27; and (3) a cellulose binding module (CBM) from about amino acid 503 to about amino acid 535 of SEQ ID NO: 27.
- the domains of T include, without limitation: (1) a signal sequence, from amino acid 1 to 33 of SEQ ID NO: 27; (2) a catalytic domain (CD) from about amino acid 41 to about amino acid 465 of SEQ ID NO: 27; and (3) a cellulose binding module (CBM) from about amino acid 503 to about amino acid 535 of SEQ ID NO: 27.
- CD catalytic domain
- CBM cellulose binding module
- reesei Cbh 2 include, without limitation: (1) a signal sequence, from amino acid 1 to 33 of SEQ ID NO: 27; (2) a catalytic domain (CD) from about amino acid 145 to about amino acid 458 of SEQ ID NO: 27; and (3) a cellulose binding module (CBM) from about amino acid 52 to about amino acid 83 of SEQ ID NO: 27.
- a signal sequence from amino acid 1 to 33 of SEQ ID NO: 27
- CD catalytic domain
- CBM cellulose binding module
- the present invention also encompasses an isolated polynucleotide comprising a nucleic acid that is at least about 70%, 75%, or 80% identical, at least about 90% to about 95% identical, or at least about 96%, 97%, 98%, 99% or 100% identical to a nucleic acid encoding a T. emersonii, H. grisea, T. aurantiacus, C. lucknowense or T. reesei Cbhl or Cbh2 domain, as described above.
- the present invention also encompasses variants of the cellulase genes, as described above.
- Variants may contain alterations in the coding regions, non-coding regions, or both. Examples are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide.
- nucleotide variants are produced by silent substitutions due to the degeneracy of the genetic code.
- acinaciformis M. darwinensis, N. walkeri, S. fibuligera, C. luckowense R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H.
- the present invention also encompasses an isolated polynucleotide encoding a fusion protein.
- the nucleic acid encoding a fusion protein comprises a first polynucleotide encoding for a T. emersonii cbhl , H. grisea cbhl, T. aurantiacusi cbhl or T. emersonii cbhl and a second polynucleotide encoding for the CBM domain of T. reesei cbhl or T. reesei cbh2 or C. lucknowense cbh2b.
- the first polynucleotide encodes T. emersonii cbhl and the second polynucleotide encodes for a CBM from T. reesei Cbhl or Cbh2.
- the first and second polynucleotides are in the same orientation, or the second polynucleotide is in the reverse orientation of the first polynucleotide.
- the first polynucleotide encodes a polypeptide that is either N-terminal or C-terminal to the polypeptide encoded by the second polynucleotide.
- the first polynucleotide and/or the second polynucleotide are encoded by codon-optimized polynucleotides, for example, polynucleotides codon-optimized for S. cerevisiae, Kluyveromyces or for both S.
- the first polynucleotide is a codon-optimized T. emersonii cbhl and the second polynucleotide encodes for a codon-optimized CBM from T. reesei Cbhl or Cbh2.
- allelic variants, orthologs, and/or species homologs are also provided in the present invention. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to any of SEQ ID NOs: 1-20, using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
- nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the particular polypeptide, In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
- the query sequence may be an entire sequence shown of any of SEQ E) NOs: 1-20, or any fragment or domain specified as described herein.
- nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence or polypeptide of the present invention can be determined conventionally using known computer programs.
- a method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. (1990) 6:237-245.) In a sequence alignment the query and subject sequences are both DNA sequences.
- RNA sequence can be compared by converting U's to T's.
- the result of said global sequence alignment is in percent identity.
- the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment.
- This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
- This corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
- a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity.
- the deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end.
- the 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%.
- a 90 base subject sequence is compared with a 100 base query sequence.
- deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query.
- percent identity calculated by FASTDB is not manually corrected.
- bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.
- nucleic acid molecule comprising at least 10, 20, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, or 800 consecutive nucleotides or more of any of SEQ ID NOs: 1-20, or domains, fragments, variants, or derivatives thereof.
- the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
- the DNA may be double stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand.
- the coding sequence which encodes the mature polypeptide can be identical to the coding sequence encoding SEQ ID NO :21-40, 46, or 52-56, or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of any one of SEQ ID NOs:21-40, 46, or 52-56.
- the present invention provides an isolated polynucleotide comprising a nucleic acid fragment which encodes at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 95, or at least 100 or more contiguous amino acids of SEQ ID NOs: 21-40, 46, or 52-56.
- 46, or 52-56 may include: only the coding sequence for the mature polypeptide; the coding sequence of any domain of the mature polypeptide; and the coding sequence for the mature polypeptide (or domain-encoding sequence) together with non coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
- polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only sequences encoding for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences.
- nucleic acid molecules having sequences at least about 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences disclosed herein encode a polypeptide having cellobiohydrolase (“Cbh”), endoglucanase (“Eg”) or beta-gluconase (“BgI”) functional activity.
- Cbh cellobiohydrolase
- Eg endoglucanase
- BgI beta-gluconase
- a polypeptide having Cbh, Eg or BgI functional activity is intended polypeptides exhibiting activity similar, but not necessarily identical, to a functional activity of the Cbh, Eg or BgI polypeptides of the present invention, as measured, for example, in a particular biological assay.
- a Cbh, Eg or BgI functional activity can routinely be measured by determining the ability of a Cbh, Eg or BgI polypeptide to hydrolyze cellulose, or by measuring the level of Cbh, Eg or BgI activity.
- nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of any of SEQ ID NOs: 1-20, or fragments thereof, will encode polypeptides having Cbh, Eg or BgI functional activity.
- degenerate variants of any of these nucleotide sequences all encode the same polypeptide, in many instances, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having Cbh, Eg or BgI functional activity.
- the polynucleotides of the present invention also comprise nucleic acids encoding a H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. luckowense R.
- speratus Thermobfida fusca, Clostridium thermocellum, Clostridium cellulolyticum, Clostrid ⁇ m josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H.
- the marker sequence may be a yeast selectable marker selected from the group consisting of URA3, HIS3, LEU2, TRPl, LYS2 or ADE2.
- a yeast selectable marker selected from the group consisting of URA3, HIS3, LEU2, TRPl, LYS2 or ADE2.
- Casey, G.P. et al. "A convenient dominant selection marker for gene transfer in industrial strains of Saccharomyces yeast: SMRl encoded resistance to the herbicide sulfometuron methyl," J. Inst. Brew. 94:93-97 (1988). Codon Optimized Polynucleotides
- the polynucleotides encoding heterologous cellulases can be codon-optimized.
- codon- optimized coding region means a nucleic acid coding region that has been adapted for expression in the cells of a given organism by replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that organism.
- CAI codon adaptation index
- DNA sequence can be checked for direct repeats, inverted repeats and mirror repeats with lengths of ten bases or longer, which can be modified manually by replacing codons with "second best" codons, i.e., codons that occur at the second highest frequency within the particular organism for which the sequence is being optimized.
- Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The "genetic code” which shows which codons encode which amino acids is reproduced herein as Table 2. As a result, many amino acids are designated by more than one codon.
- amino acids alanine and proline are coded for by four triplets, serine and arginine by six, whereas tryptophan and methionine are coded by just one triplet.
- This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA.
- Codon preference or codon bias differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules.
- mRNA messenger RNA
- tRNA transfer RNA
- the predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.
- Codon usage tables are readily available, for example, at http://phenotype.biosci.umbc.edu/codon/sgd/index.php (visited May 7, 2008) or at http://www.kazusa.or.jp/codon/ (visited March 20, 2008), and these tables can be adapted in a number of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000).
- Codon usage tables for yeast are reproduced below as Table 3. This table uses mRNA nomenclature, and so instead of thymine (T) which is found in DNA, the tables use uracil (U) which is found in RNA. The Table has been adapted so that frequencies are calculated for each amino acid, rather than for all 64 codons. TABLE 3: Codon Usage Table for Saccharomyces cerevisiae Genes
- Codon-optimized coding regions can be designed by various different methods.
- a codon usage table is used to find the single most frequent codon used for any given amino acid, and that codon is used each time that particular amino acid appears in the polypeptide sequence. For example, referring to Table 3 above, for leucine, the most frequent codon is UUG, which is used 27.2% of the time. Thus all the leucine residues in a given amino acid sequence would be assigned the codon UUG.
- the actual frequencies of the codons are distributed randomly throughout the coding sequence.
- a hypothetical polypeptide sequence had 100 leucine residues, referring to Table 3 for frequency of usage in the S. cerevisiae, about 5, or 5% of the leucine codons would be CUC, about 11, or 11% of the leucine codons would be CUG, about 12, or 12% of the leucine codons would be CUU, about 13, or 13% of the leucine codons would be CUA, about 26, or 26% of the leucine codons would be UUA, and about 27, or 27% of the leucine codons would be UUG.
- the term “about” is used precisely to account for fractional percentages of codon frequencies for a given amino acid.
- “about” is defined as one amino acid more or one amino acid less than the value given. The whole number value of amino acids is rounded up if the fractional frequency of usage is 0.50 or greater, and is rounded down if the fractional frequency of use is 0.49 or less.
- the fractional frequency of codon usage would be calculated by multiplying 62 by the frequencies for the various codons.
- 7.28 percent of 62 equals 4.51 UUA codons, or "about 5,” i.e., 4, 5, or 6 UUA codons, 12.66 percent of 62 equals 7.85 UUG codons or "about 8," i.e., 7, 8, or 9 UUG codons, 12.87 percent of 62 equals 7.98 CUU codons, or "about 8," i.e., 7, 8, or 9 CUU codons, 19.56 percent of 62 equals 12.13 CUC codons or "about 12,” i.e., 11, 12, or 13 CUC codons, 7.00 percent of 62 equals 4.34 CUA codons or "about 4," i.e., 3, 4, or 5 CUA codons, and 40.62 percent of 62 equals 25.19 CUG codons, or "about 25,” i.e., 24, 25, or 26 CUG codons.
- Randomly assigning codons at an optimized frequency to encode a given polypeptide sequence can be done manually by calculating codon frequencies for each amino acid, and then assigning the codons to the polypeptide sequence randomly.
- various algorithms and computer software programs are readily available to those of ordinary skill in the art. For example, the "EditSeq” function in the Lasergene Package, available from DNAstar, Inc., Madison, WI, the backtranslation function in the VectorNTI Suite, available from InforMax, Inc., Bethesda, MD, and the "backtranslate” function in the GCG-- Wisconsin Package, available from Accelrys, Inc., San Diego, CA.
- a number of options are available for synthesizing codon optimized coding regions designed by any of the methods described above, using standard and routine molecular biological manipulations well known to those of ordinary skill in the art.
- a series of complementary oligonucleotide pairs of 80-90 nucleotides each in length and spanning the length of the desired sequence is synthesized by standard methods.
- oligonucleotide pairs are synthesized such that upon annealing, they form double stranded fragments of 80-90 base pairs, containing cohesive ends, e.g., each oligonucleotide in the pair is synthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond the region that is complementary to the other oligonucleotide in the pair.
- the single-stranded ends of each pair of oligonucleotides is designed to anneal with the single-stranded end of another pair of oligonucleotides.
- the oligonucleotide pairs are allowed to anneal, and approximately five to six of these double-stranded fragments are then allowed to anneal together via the cohesive single stranded ends, and then they ligated together and cloned into a standard bacterial cloning vector, for example, a TOPO vector available from Invitrogen Corporation, Carlsbad, CA.
- the construct is then sequenced by standard methods. Several of these constructs consisting of 5 to 6 fragments of 80 to 90 base pair fragments ligated together, i.e., fragments of about 500 base pairs, are prepared, such that the entire desired sequence is represented in a series of plasmid constructs.
- an entire polypeptide sequence, or fragment, variant, or derivative thereof is codon optimized by any of the methods described herein.
- Various desired fragments, variants or derivatives are designed, and each is then codon-optimized individually,
- partially codon-optimized coding regions of the present invention can be designed and constructed.
- the invention includes a nucleic acid fragment of a codon-optimized coding region encoding a polypeptide in which at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the codon positions have been codon-optimized for a given species. That is, they contain a codon that is preferentially used in the genes of a desired species, e.g., a yeast species such as Saccharomyces cerevisiae or Kluveromyces, in place of a codon that is normally used in the native nucleic acid sequence.
- a desired species e.g., a yeast species such as Saccharomyces cerevisiae or Kluveromyces
- a full-length polypeptide sequence is codon-optimized for a given species resulting in a codon-optimized coding region encoding the entire polypeptide, and then nucleic acid fragments of the codon-optimized coding region, which encode fragments, variants, and derivatives of the polypeptide are made from the original codon-optimized coding region.
- nucleic acid fragments encoding fragments, variants, and derivatives would not necessarily be fully codon optimized for the given species.
- the codon-optimized coding regions can be, for example, versions encoding a cellobiohydrolase, endoglucanase or beta-glucosidase from H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. luckowense R.
- Clostridum thermocellum Clostridium cellulolyticum
- Clostridum josui Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H.
- Codon optimization is carried out for a particular species by methods described herein, for example, in certain embodiments codon-optimized coding regions encoding polypeptides of H. grisea, T. aurantiacus, T. emersonii, T.
- yeast codon usage e.g., Saccharomyces cerevisiae, Kluyveromyces lactis and/or Kluyveromyces marxianus.
- polynucleotides, vectors, and other expression constructs comprising codon-optimized coding regions encoding polypeptides of H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. luckowense R.
- Clostridum thermocellum Clostridium cellulolyticum
- Clostridum josui Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum, Aspergillus kawachii, Heterodera schachtii, H.
- a codon-optimized coding region encoding any of SEQ ID NOs:21-40, 46, or 52-56 or domain, fragment, variant, or derivative thereof, is optimized according to codon usage in yeast (Saccharomyces cerevisiae, Kluyveromyces lactis or Kluyveromyces marxianus).
- the sequences are codon-optimized specifically for expression in Saccharomyces cerevisiae.
- the sequences are codon-optimized for expression in Kluyveromyces.
- a sequence is simultaneously codon-optimized for optimal expression in both Saccharomyces cerevisiae and in Kluyveromyces.
- a codon-optimized coding region encoding any of SEQ E) NOs: 21-40, 46, or 52-56 may be optimized according to codon usage in any plant, animal, or microbial species.
- the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
- Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
- the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
- the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the present invention.
- the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
- the polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques.
- the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide.
- Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; and yeast plasmids.
- any other vector may be used as long as it is replicable and viable in the host.
- the appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
- the DNA sequence in the expression vector is operatively associated with an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
- promoter an appropriate expression control sequence(s) to direct mRNA synthesis.
- promoters are as follows:
- promoter sequences from stress and starvation response genes are useful in the present invention.
- ATGIl ATGIl, ATG12, ATG13, ATG14, ATG15, ATGl 6, ATGl 7, ATGl 8, and ATGl 9 may be used. Any suitable promoter to drive gene expression in the host cells of the invention may be used. Additionally the E. coli, lac or trp, and other promoters known to control expression of genes in prokaryotic or lower eukaryotic cells can be used.
- the expression vectors may contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as URA3, HIS3, LEU2, TRPl, LYS2 or ADE2, dihydrofolate reductase, neomycin (G418) resistance or zeocin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli.
- selectable marker genes such as URA3, HIS3, LEU2, TRPl, LYS2 or ADE2, dihydrofolate reductase, neomycin (G418) resistance or zeocin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli.
- the expression vector may also contain a ribosome binding site for translation initiation and/or a transcription terminator.
- the vector may also include appropriate sequences for amplifying expression, or may include additional regulatory regions.
- the vector containing the appropriate DNA sequence as herein, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
- the present invention relates to host cells containing the above-described constructs.
- the host cell can be a host cell as described elsewhere in the application.
- the host cell can be, for example, a lower eukaryotic cell, such as a yeast cell, e.g., Saccharomyces cerevisiae or Kluyveromyces, or the host cell can be a prokaryotic cell, such as a bacterial cell.
- bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium
- thermophilic or mesophlic bacteria such as E. coli, Streptomyces, Salmonella typhimurium
- fungal cells such as yeast
- plant cells etc.
- the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
- yeast is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schwanniomyces occidentalis, Issatchenkia orientalis, Kluyveromyces marxianus, Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomces, Pythium, Rhodosporidium, Rhodotorula, Trichosporon and Yarrowia.
- the present invention is also directed to use of host cells and co-cultures to produce ethanol from cellulosic substrates. Such methods can be accomplished, for example, by contacting a cellulosic substrate with a host cell or a co-culture of the present invention.
- suitable lignocellulosic material may be any feedstock that contains soluble and/or insoluble cellulose, where the insoluble cellulose may be in a crystalline or non-crystalline form.
- the lignocellulosic biomass comprises, for example, wood, corn, corn stover, sawdust, bark, leaves, agricultural and forestry residues, grasses such as switchgrass, ruminant digestion products, municipal wastes, paper mill effluent, newspaper, cardboard or combinations thereof.
- the invention is directed to a method for hydrolyzing a cellulosic substrate, for example a cellulosic substrate as described above, by contacting the cellulosic substrate with a host cell of the invention, In some embodiments, the invention is directed to a method for hydrolyzing a cellulosic substrate, for example a cellulosic substrate as described above, by contacting the cellulosic substrate with a co- culture comprising yeast cells expressing heterologous cellulases.
- the invention is directed to a method for fermenting cellulose. Such methods can be accomplished, for example, by culturing a host cell or co- culture in a medium that contains insoluble cellulose to allow saccharification and fermentation of the cellulose.
- the production of ethanol can, according to the present invention, be performed at temperatures of at least about 30° C, about 31° C, about 32° C, about 33° C, about 34° C, about 35° C, about 36° C, about 37° C, about 38° C, about 39° C, about 40° C, about 41° C, about 42° C, about 43 °C, about 44 °C, about 45 °C, about 46 °C, about 47 °C, about 48 °C, about 49 °C, or about 50° C.
- thermotolerant host cell can produce ethanol from cellulose at temperatures above about 30° C, about 31° C, about 32° C, about 33° C, about 34° C, about 35° C, about 36° C, about 37° C, about 38° C, about 39° C, about 40° C, about 41° C, about 42° C, or about 43 °C, or about 44 °C, or about 45 °C, or about 50° C.
- the thermotolterant host cell can produce ethanol from cellulose at temperatures from about 30° C to 60° C, about 30° C to 55° C, about 30° C to 50° C, about 40° C to 60° C, about 40° C to 55° C or about 40° C to 50° C.
- methods of producing ethanol can comprise contacting a cellulosic substrate with a host cell or co-culture of the invention and additionally contacting the cellulosic substrate with externally produced cellulase enzymes.
- Exemplary externally produced cellulase enzymes are commercially available and are known to those of skill in the art.
- the invention is also directed to methods of reducing the amount of externally produced cellulase enzymes required to produce a given amount of ethanol from cellulose comprising contacting the cellulose with externally produced cellulases and with a host cell or co-culture of the invention.
- the same amount of ethanol production can be achieved using at least about 5%, 10%, 15%, 20%, 25%, 30%, or 50% less externally produced cellulases.
- no external cellulase is added, or less than about 5% of the cellulase is externally added cellulase, or less than about 10% of the cellulase is externally added cellulase, or less than about 15% of the cellulase is externally added cellulase.
- the methods comprise producing ethanol at a particular rate.
- ethanol is produced at a rate of at least about 0.1 mg per hour per liter, at least about 0.25 mg per hour per liter, at least about 0.5 mg per hour per liter, at least about 0.75 mg per hour per liter, at least about 1.0 mg per hour per liter, at least about 2.0 mg per hour per liter, at least about 5.0 mg per hour per liter, at least about 10 mg per hour per liter, at least about 15 mg per hour per liter, at least about 20.0 mg per hour per liter, at least about 25 mg per hour per liter, at least about 30 mg per hour per liter, at least about 50 mg per hour per liter, at least about 100 mg per hour per liter, at least about 200 mg per hour per liter, at least about 300 mg per hour per liter, at least about 400 mg per hour per liter, or at least about 500 mg per hour per liter.
- the host cells of the present invention can produce ethanol at a rate of at least about 0.1 mg per hour per liter, at least about 0.25 mg per hour per liter, at least about 0.5 mg per hour per liter, at least about 0.75 mg per hour per liter, at least about 1.0 mg per hour per liter, at least about 2.0 mg per hour per liter, at least about 5.0 mg per hour per liter, at least about 10 mg per hour per liter, at least about 15 mg per hour per liter, at least about 20.0 mg per hour per liter, at least about 25 mg per hour per liter, at least about 30 mg per hour per liter, at least about 50 mg per hour per liter, at least about 100 mg per hour per liter, at least about 200 mg per hour per liter, at least about 300 mg per hour per liter, at least about 400 mg per hour per liter, or at least about 500 mg per hour per liter more than a control strain (lacking heterologous cellulases) and grown under the same conditions.
- a control strain lacking hetero
- Ethanol production can be measured using any method known in the art. For example, the quantity of ethanol in fermentation samples can be assessed using HPLC analysis. Many ethanol assay kits are commercially available that use, for example, alcohol oxidase enzyme based assays. Methods of determining ethanol production are within the scope of those skilled in the art from the teachings herein.
- the present invention presents a number of important steps forward for creating a yeast capable of consolidated bioprocessing. It describes improved cellulolytic yeast created by expressing combinations of heterologous cellulases.
- the present invention demonstrates for the first time, the ability of transformed Kluyveromyces to produce ethanol from cellulose, the ability of yeast strains expressing only secreted heterologous cellulases to produce ethanol from cellulose, and the ability of co-cultures of multiple yeast strains expressing different cellulases to produce ethanol from cellulose.
- yeast strains and co-cultures of yeast strains can increase the efficiency of simultaneous saccharification and fermentation (SSF) processes.
- SSF simultaneous saccharification and fermentation
- Biolabs was used for plasmid transformation and propagation.
- Cells were grown in LB medium (5 g/L yeast extract, 5 g/L NaCl, 10 g/L tryptone) supplemented with ampicillin (100 mg/L), kanamycin (50mg/L), or zeocin (20 mg/L).
- LB was adjusted to pH 7.0.
- 15 g/L agar was added when solid media was desired.
- Yeast strains were routinely grown in YPD (10 g/L yeast extract, 20 g/L peptone,
- YPC 10 g/L yeast extract, 20 g/L peptone, 20 g/L cellobiose
- YNB + glucose 6.7 g/L Yeast Nitrogen Base without amino acids, and supplemented with appropriate amino acids for strain, 20 g/L glucose
- G418 250 mg/L unless specified
- zeocin 20 mg/L unless specified
- PCR was performed using Phusion polymerase (New England Biolabs) for cloning, and Taq polymerase (New England Biolabs) for screening transformants, and in some cases Advantage Polymerase (Clontech) for PCR of genes for correcting auxotrophies. Manufacturers guidelines were followed as supplied. Restriction enzymes were purchased from New England Biolabs and digests were set up according to the supplied guidelines. Ligations were performed using the Quick ligation kit (New England Biolabs) as specified by the manufacturer. Gel purification was performed using either Qiagen or Zymo research kits, PCR product and digest purifications were performed using Zymo research kits, and Qiagen midi and miniprep kits were used for purification of plasmid DNA.
- Yeast mediated ligation was used to create some constructs (Ma et al. Gene 55:201-216 (1987)). This was done by creating DNA fragments to be cloned with 20-40bp of homology with the other pieces to be combined and/or the backbone vector.
- a backbone vector (pRS426), able to replicate in yeast, and with the Ura3 gene for selection, was then transformed into yeast by standard methods with the target sequences for cloning. Transformed yeast recombine these fragments to form a whole construct and the resulting plasmid allows selection on media without uracil.
- Table 4 Table 4, and the primers used in vector construction are shown in Table 5. Table 4. Plasmids used.
- the yeast expression vector YEpENO-BBH was created to facilitate heterologous expression under control of the S. cerevisiae enolase 1 (ENOl) gene promoter and terminator.
- the vector was also useful because the expression cassette from this vector could be simply excised using a BamHI, BgIII digest.
- YEpENOl (Den Haan et al, Metabolic Engineering. 9: 87-942007) contains the YEp352 backbone with the ENOl gene promoter and terminator sequences cloned into the BamHI and HindIII sites. This plasmid was digested with BamHI and the overhang filled in with Klenow polymerase and dNTPs to remove the BamHI site.
- YEpENO-BBHtemplate was used as template for a PCR reaction with primers ENOBB-left (5'-
- GenScript Corporation were cloned onto the plasmid pUC57.
- the resulting vectors were digested with EcoRI and Xhol to excise the cbh genes which were subsequently cloned into an EcoRI and Xhol digested YEpENO-BBH. This created the plasmids pRDH103
- Tecbhl with the cbh encoding genes under transcriptional control of the ENOl promoter and terminator.
- pRDHlOl was created to express the T. reesei CBHl from pBZD_10631_20641.
- Takara ExTaq enzyme was used as directed and to amplify the sTrcbhl from pBZD_10631_20641 using primers sCBHl/2 L and sCBHIR. The fragment was then isolated and digested with EcoRI and Xhol.
- YEpENO-BBH was also digested with EcoRI and Xhol and the relevant bands were isolated and ligated.
- This plasmid was designated pRDH107.
- pRDH109 contains the same expression cassettes as pRDH108 but in pRDH108 the gene expression cassettes are in the reverse orientation relative to each other.
- T. emersonii CBHl with a c-terminal fusion of the CBM of T. reesei CBHl were also created.
- Table 5 lists the oligonucleotides used for these constructs.
- a PCR product was amplified with the oligonucleotides 395 Te cbhl Syntl PacI-ATG and 398 Te cbhl synt core Smal using pRDH105 as the template, digested with PmII and Smal and the 800 bp fragment was isolated.
- a second PCR product was amplified with oligonucleotides 399 Trcbhl synt CBM5 Mly1HincII and 400 Trcbhl synt CBM AscIXhoI with pRDHlOl as the template, digested with MIyI and Xhol and the 180 bp fragment was isolated. The two PCR fragments were ligated with the 6.9 kb Pmll-Xhol fragment of pRDH105 resulting in pMU624.
- the PGKl promoter was amplified with primers 379 ScPGKlprom -786 Sacl+Apal and 380 ScPGKl prom EcoRI-PacI and pAJ410 as the template and digested with Pad and EcoRI. The T.
- reesei cbh2 ORF was amplified from pTTcOl (Teeri et al., Gene 51:43-52, 1987) with oligonucleotides 381 CBH2 WT EcoRI-PacI-ATG and 386 CBH2 WT TAA- Ascl- EcoRI, digested with Pad and EcoRI, and ligated with the SacI-EcoRI digested pAJ401 resulting in pMI508.
- the Pad- Ascl fragment in pMI508 was replaced by a synthetic 1.4 kb T. reesei egll gene resulting in pMI522.
- pMI522 The 1.9 kb fragment of pMI522 was digested with PmII and Xhol and ligated to the 6.4 kb Pmll-Xhol fragment of pRDH107 resulting in pMI568.
- pMI568 was digested with Pad and Ascl and the 7 kb fragment was ligated to the 1.5 kb fragment of pMI558 producing pMU784 for the expression of C. lucknowense cbh2b.
- a set of 2-micron vectors was also constructed for the expression of endoglucanases in S. cerevisiae, as well as related plasmids to act as controls.
- pMU451 was created as a control vector and for cloning the cellulases under control of the ENOl promoter and terminator. This was done by adding a Pacl/Ascl linker into the EcoRI/XhoI site of pMU451. Synthetic genes ordered from Codon Devices and received in pUC57 were cloned into this vector as Pacl/Ascl fragments.
- Vectors created this way and listed in Table 4 are: pMU458, pMU463, pMU465, pMU469, pMU471, pMU472, pMU473, pMU475, pMU499, pMU500, and pMU5O3.
- Vectors for integrating secreted versions of cellulases at the delta integration sites in S. cerevisiae, or for integration into the genome of K. marxianus were created from the pBKD l and ⁇ BKD_2 constructs.
- the S.fibuligera BGLl (SfBGLI) was cloned by PCR from ySFI (van Rooyen et al., J. Biotechnol. 120: 284-95 (2005)).
- the endoglucanase (TrEGI) used was the sequence give in Table 1.
- the cellulase encoding genes were cloned via PCR (using Pad and Ascl sites) into pBKD l and pBKD_2 - to create pBKDl-BGLl and pBKD2-sEGl.
- the ENOlP-sEGl-ENOlT cassette from pBKD2- sEGl was subsequently sub cloned as a Spel, Notl fragment to pBKDl-BGLl to create pBKDl-BGLl-sEGl.
- pMU562 used for integrating cellulases into K. marxianus, was generated by cutting with pMU185 (pUG66) with Notl and isolating a 1190 bp lox P ZeoR containing insert. This insert was ligated into a Notl digested 4.5Kb delta-integration vector to produce pMU562.
- pMU576 was generated by cutting T. reesei CBH2 containing plasmid pMU291 with Ascl /Pad, isolating a 1491 bp CBH2 gene and Ii gating it into delta- integration vector pMU562 cut with Ascl/Pacl.
- pMU577 was generated by cutting T.
- emersonii CBHl from ⁇ MU398 with Ascl/ Pacl, isolating a 1380 bp CBHl gene and ligating into delta-integration vector pMU562 cut with Ascl/Pacl.
- a set of recombinant cellulase constructs (pMU661 to pMU668 and pMU750, pMU755, pMU809 — see Table 4), including a variety of endoglucanases and cellobiohydrolases, was incorporated into pMU562 for co-transformation.
- S. cerevisiae cells for transformation were prepared by growing to saturation in
- electroporation buffer IM sorbitol, 2OmM HEPES
- resuspended in 267 ⁇ L electroporation buffer The same protocol was used for transforming K. lactis and K. marxianus strains, except that 50 mLs of YPD was inoculated with 0.5 mL from an overnight culture, grown for 4 hours at 37 °C, and then centrifuged and prepared as above. Additionally, incubations and recovery steps were carried out at 37 °C.
- yeast strains listed in Table 6 were created using the vectors and transformation protocols as described.
- Table 6 Yeast Strains.
- the plasmid pBKDl-BGLl-sEGl (pMU276) was digested with Accl and transformed to S. cerevisiae Y294 by electrotransformation to create a strain with delta integrated copies of the SfBGLI and TrEGI, designated M0243. Episomal plasmids were then transformed to S. cerevisiae Y294 and/or M0243.
- M0282 was created by transforming M0248 with Accl digested pBKDl -BGLI- sEGI, as described above, except that the transformation mixture was spread on plated containing 10 g/L BMCC with lOg/L yeast extract and 20 g/L peptone.
- BMCC Bacterial microcrystalline cellulose
- BMCC as received was stirred CVN at 4C in water. After the substrate was rehydrated, it was washed 6 times with water and resuspended in water. The dry weight of the substrate was measured by drying samples at 105 C until constant weight was obtained.
- Phosphoric acid swollen cellulose was prepared as in Zhang and Lynd
- ⁇ -glucosidase activity was measured in a manner similar to McBride, J.E., et al.,
- yeast strains were grown to saturation in YPD or YPC media with or without appropriate antibiotics, the optical density at 600nm (OD(600)) was measured, and an 0.5 mL sample of the cultures was taken. This sample was centrifuged, the supernatant was separated and saved, and the cell pellet was washed 2X 50 mM citrate buffer, pH 5.0.
- Reactions for supernatants were made up of 50 ⁇ L sample, 50 ⁇ L citrate buffer, and 50 ⁇ L 20 mM p-nitrophenyl- ⁇ - D-glucopyranoside (PNPG) substrate.
- Reactions with washed cells consisted of 25 ⁇ L of cells, 75 ⁇ L citrate buffer, and 50 ⁇ L PNPG substrate. If the activity was too high for the range of the standard curve, a lower cell concentration was used and the assay was re-run.
- the standard curve consisted of a 2-fold dilution series of nitrophenol (PNP) standards, starting at 500 nM, and ending at 7.8 nM, and a buffer blank was included.
- the microtiter plate was incubated at 37 °C for 10 minutes along with the reaction substrate.
- the reaction was carried out by adding the substrate, incubating for 30 min., and stopping with 150 ⁇ L of 2M Na 2 CO 3 .
- the plate was then centrifuged at 2500 rpm for 5 minutes, and 150 ⁇ L of supernatant was transferred to another plate. The absorbance at 405 nm was read for each well.
- Endoglucanase activity was qualitatively detected by observing clearing zones on synthetic complete media (as above, but including 20 g/L glucose) plates with 0.1% carboxymethyl cellulose (CMC) stained with congo red (Beguin, Anal. Biochem. 131: 333-6 (1983)). Cells were grown for 2-3 days on the plates and were washed off the plate with IM Tris-HCL buffer pH 7.5. The plates were then stained for 10 minutes with a 0.1% Congo red solution, and extra dye was subsequently washed off with IM NaCl.
- CMC carboxymethyl cellulose
- CBHl activity was detected using the substrate 4-Methylumbelliferyl- ⁇ -D- lactoside (MULac). Assays were carried out by mixing 50 ⁇ L of yeast supernatant with 50 ⁇ L of a 4mM MUlac substrate solution made in 50 mM citrate buffer pH 5.5. The reaction was allowed to proceed for 30 minutes and then stopped with IM Na 2 CO 3 . The fluorescence in each well was read in a microtiter plate reader (ex. 355 nm and em. 460 nm).
- Enzyme activity on PASC and Avicel were measured using the protocol described in Den Haan et al, Enzyme and Microbial Technology 40: 1291-1299 (2007). Briefly, yeast supernatants were incubated with cellulose at 4 °C to bind the cellulase. The cellulose was then filtered from the yeast supernatant, resuspended in citrate buffer and sodium azide, and incubated at 37 °C. Accumulation of sugar was measured in the reaction by sampling and performing a phenol-sulfuric acid assay. (See Example 10 and Table 9.)
- Example 2 Strains to be tested were grown in YPD in deep-well 96 well plates at 35°C with shaking at 900 RPM. After growing, plates were centrifuged at 4000 rpm for 10 min. 300 ⁇ L substrate (2% avicel, 5OmM sodium acetate buffer, 0.02% sodium azide, ⁇ - glucosidase — l ⁇ L per mL) was added to a new 96-well deep well plate, without allowing the avicel to settle. 300 ⁇ L of yeast supernatant was added to this substrate, and 100 ⁇ L was taken for an initial sample. The assay plate is incubated at 35°C, with shaking at 800 rpm, and samples were taken at 24 and 48 hours.
- substrate 2% avicel, 5OmM sodium acetate buffer, 0.02% sodium azide, ⁇ - glucosidase — l ⁇ L per mL
- 300 ⁇ L of yeast supernatant was added to this substrate, and 100 ⁇ L
- Example 1 Production of Kluyveromyces expressing heterologous ⁇ -glucosidase and endoglucanase
- Kluyveromyces marxianus ATCC strain #10606; MOl 57
- Kluyveromyces lactis ATCC strain # 34440
- Vectors containing yeast delta integration sequences, the KanMX marker and sequences encoding S.f. BGLI and T.r. EGI were transformed into Kluyveromyces according to the yeast transformation protocol as described above, and selected on G418. Transformants were verified by PCR and then tested by CMC assay. The results are shown in Figure 1. The presence of the heterologous cellulase activity is indicated by a clearing zone on the CMC plate. As shown in Figure 1, neither an untransformed K. lactis strain (colony 8) or an untransformed K. marxianus strain (colony 16) showed endoglucanase activity. However, 6 of 7 transformed K. lactis colonies showed CMCase activity, and all 7 transformed K. marxianus colonies showed CMCase activity. MO413 and MO414 were identified as two K. marxianus colonies showing CMCase activity.
- K. marxianus (MOl 57) was transformed with constructs containing T. reesei CBH2, T. emersonii CBHl or both.
- MO414 K marxianus transformed with S.f. BGLI and T.r. EGI was transformed with constructs containing T . reesei CBH2, T. emersonii CBHl or both.
- Transformations were performed as described in above. CBHl activity was then detected using the substrate 4-Methylumbelliferyl- ⁇ -D-lactoside (MU-Lac) as described above. The assay was performed on eight colonies of each transformant and the three colonies showing the highest activity were averaged. The results are shown in Figure 2 and demonstrate that strains transformed with T. emersonii CBHl had high MU-lac activity.
- Example 3 Production of Kluyveromyces expressing a library of cellulases
- Kluveromyces strains were also created by transforming yeast with a library of cellulases (creation of library was described above). For example, MO413 was transformed with a library of cellulases containing a zeocin marker to produce novel strains MO601-MO604 and MO611-MO617. In addition, MO157 (K. marxianus) was transformed with the same library and novel strains MO618-MO625 were identified. Activity on Avicel was assessed at 48 hours as described above, and the results, shown in Figure 3, demonstrate that Kluveryomces transformed with a library of heterologous cellulases also have Avicelase activity at 35° C. Tranformants of MO157 with the library showed the highest activity. Avicelase activity at 45° C was also demonstrated (data not shown).
- Example 5 Production of S. cerevisiae expressing heterologous cellulases
- BMCC media containing bacterial microcrystalline cellulose
- T. emersonii CBHl and T. reesei CBH2 were transformed with a construct allowing T. reesei EGI and S. fibuligera BGLI expression (pKD-BGLI-sEGI). That transformation was plated on a BMCC solid agar plate and five colonies appeared on the plate after seven days (data not shown). Yeast from the largest of the five colonies was isolated as strain MO282. (MO282 is described in more detail above.) The three control strains were tested for growth on the same plates. One strain expressed with T. emersonii CBHI and T. reesei CBH2, and two strains expressed T. reesei EGI and S. fibuligera BGLI. No colonies appeared on plates with control yeast strains (data not shown).
- Figure 5 shows that MO282, which expresses all 4 secreted cellulases grew to a much greater extent on BMCC than a plasmid only control (MO249), a strain expressing only T. emersonii CBHl and T. reesei CBH2 (MO249), and a strain expressing 4 tethered cellulases (MO144).
- Example 6 S. cerevisiae expressing heterologous cellulases can produce ethanol from Avicel and Pretreated Hardwood
- Avicel media was made using the non-glucose components of synthetic complete medium for yeast including, yeast nitrogen base without amino acids — 6.7 g/L, and supplemented with a complete amino acid mix (complete supplemental mixture).
- yeast extract (10 g/L) and peptone (20 g/L) (YP) were used as supplements in growth experiments.
- Cultivation conditions were anaerobic and were maintained by flushing sealed glass bottles with N2 after carbon source addition and before autoclaving.
- Non-carbon media components were added as 1OX solutions by filter sterilizing after autoclaving. Inoculation into Avicel cultures was done at 20% by volume. Quantification of ethanol in fermentation samples was carried out by HPLC analysis, and initial ethanol concentrations in bottles (from precultures) was subtracted from all subsequent data points.
- EGI, T. reesei CBH2, and T. emersonii CBHl was able to produce ethanol directly from avicel PH 105 as compared to the control strain (M0249) when YNB media components were used.
- cellulase loading can be reduced -15% compared to the control, and for YNB media, cellulase loading can be reduced -5%.
- ethanol productivity was increased between 5 and 20% for strains expressing cellulases in YP media as compared to the control. It was increased between 10 and 20% for strains cultured in YNB media compared to the control.
- SSF processes can be improved in terms of ethanol yield from biomass and ethanol productivity if strains expressing secreted cellulases are used in combination with exogenously added cellulases.
- cellulase loadings required to achieve a particular percentage of theoretical ethanol yield can be reduced when strains expressing recombinant cellulases are added.
- Example 8 Transformed yeast strains also increase efficiency of externally added cellulases in the production of ethanol from Avicel.
- FIG. 12 presents cellulase enzyme savings based on theoretical ethanol yield at 168 hours in an SSF experiment. SSF was performed in 30 ml of nitrogen purged YP + 15% Avicel in pressure bottles. External cellulase mix at a ratio of 5 Spyzme:l Novozyme-188 was used. The experiment was continued for 168 hours and sampling was done each day for ethanol estimation by HPLC.
- the artificial protein sequence was designed as a consensus (the most common) sequence for these proteins.
- the predicted signal sequence was exchanged by S.cerevisiae alpha mating factor pre signal sequence, and the sequence of the consensus CBHl protein is shown below. Capital letters indicate the S.cerevisiae alpha mating factor pre signal sequence.
- MRFPSIFT AVLF AASSALAqqagtltaethpsltwqkctsggscttvngswidanwrwvhatsgst ncytgntwdttlcpddvtcaqncaldgadysstygvttsgnslrlnfvtqgsqknvgsrlyhneddttyqmfkllg qeftfdvdvsnlpcglngalyfvamdadggmskypgnkagakygtgycdsqcprdlkfmgqanvegweps sndanagignhgsccaemdiweansistaftphpcdtigqtmcegdscggtyssdryggtcdpdgcdfnpyr mgnktfygpgki ⁇ dttkkvtvvtqfitgssgtl
- the codon optimized sequence was inserted into the episomal yeast expression vector (pMU451) under control of ENOl promoter and terminator into Pacl/Ascl sites.
- the resulting expression constructs (pMU505) was transformed into MO375 host strain that derived from Y294 (MOO 13) in which His3 and Trpl auxotrophies were rescued by transformation with S.cerevisiae His3 and Trpl PCR products.
- the resulting strain expressing the CBHl consensus sequence was named MO429.
- Example 11 Co-cultures of yeast strains expressing different heterologous cellulases produce ethanol from Avicel
- a co-culture of a number of cellulase producing yeast strains also showed the ability to make ethanol from Avicel PH105 in YNB media (Figure 16).
- 5 strains independently producing T. emersonii CBHl (M0247), T. aurantiacus CBHl (M0266), H. grisea CBHl (M0265), a combination of T. emersonii CBHl and T. reesei CBH2 (M0248), and a combination of T. reesei EGI and S. fibuligera BGLI (M0244) were mixed in equal proportion by volume and then inoculated at 20% by volume.
- heterologously expressed cellulases in each of these strains was secreted. Media and culture conditions were as described above for Avicel experiments.
- the data in Figure 16 demonstrate that heterologous cellulases do not need to be expressed in an individual yeast strain in order to produce ethanol from cellulose. Instead, yeast strains expressing different secreted heterologous cellulases can be cultured together in order to produce ethanol from cellulose without the addition of any exogenous cellulases.
- M0566 M0424 with FUR deletion
- M0592 M0449 with FUR deletion
- M0563 (same as Y294/pMI574 furl ⁇ ): Secreted Cl CBH2b
- M0567 (same as Y294/pMI529 furl ⁇ ): Secreted TeCBHl+CBD.
- These strains were grown in liquid YPD for 3 days, until the culture was saturate for pre-culture. At this point they were used to inoculate experiments where avicel (10%) was used as the substrate, and the 4 strains were mixed at equal volume prior to inoculation.
- Figure 17 demonstrates that the co-cultured strains are capable of producing ethanol directly from avicel in the absence of any added cellulase enzyme.
- the co- culture produces about 4-fold more ethanol after 168 hours as compared to the control strain, and about 3-fold more than M0288.
- This co-culture was also used in SSF experiments where Zoomerase cellulase enzyme cocktail was used at 5 different loadings (10 mg protein/g avicel, 7.5 mg/g, 5 mg/g, and 2.5 mg/g, and 0 mg/g), and strains were inoculated at 10% by volume.
- Figure 18 presents the raw data for ethanol production at a variety of cellulase loadings by the co-culture, M0288, and M0249.
- Figure 18A shows that at all cellulase loadings tested, the co-cultured strains produced significantly more ethanol than a control not producing cellulase.
- Figure 18B shows that at all cellulase loadings tested, the co- culture produced more ethanol than the previously tested strain M0288.
- Figure 19 shows the percentage of the theoretical yield of ethanol that could be achieved with each of these cultures after 168 hours of SSF using a variety of cellulase loadings. The data demonstrate that the co-cultured strains would achieve about a 2-fold reduction in cellulase relative to the control strain, and approximately a 35% reduction compared to M0288.
- M0509 (ATCC deposit designation , deposited on November 23, 2009) is a strain of Saccharomyces cerevisiae that combines the ability to metabolize xylose with the robustness required to ferment sugars in the presence of pretreated hardwood inhibitors. M0509 was created in a three-step process. First, industrial strains of S. cerevisiae were benchmarked to identify strains possessing a level of robustness/hardiness sufficient for simultaneous saccharification and fermentation (SSF) of pretreated mixed hardwood substrates. Strain M0086, a diploid strain of strain of S. cerevisiae, satisfied this first requirement. Second, M0086 was genetically engineered with the ability to utilize xylose, resulting in strain M0407. Third, M0407 was adapted for several weeks in a chemostat containing xylose media with pretreatment inhibitors, generating strain M0509.
- SSF simultaneous saccharification and fermentation
- Strain M0407 was genetically engineered from M0086 to utilize xylose. This engineering required seven genetic modifications. The primary modification was the functional expression of the heterologous xylose isomerase gene, XyIA, isolated from the anaerobic fungus Piromyces sp. E2. The S. cerevisiae structural genes coding for all five enzymes involved in the conversion of xylulose to glycolytic intermediates were also overexpressed: xylulokinase, ribulose 5-phosphate isomerase, ribulose 5-phosphate epimerase, transketolase and transaldolase.
- XyIA heterologous xylose isomerase gene
- the GRE3 gene encoding aldose reductase was deleted to minimise xylitol production.
- the seven modified genes are listed in Figure 39.
- the genetic modifications at the GRE3, RKIl, RPEl, TALI, and TKLl loci were designed to leave behind minimal vector DNA and no antibiotic markers. Each locus' DNA was sequenced to confirm the expected results. Each of the seven genetic modifications were sequentially introduced into strain M0086.
- Figure 40 shows the progression of modifications from top to bottom together with the designations for the strain at each step in the process, starting with M0086 and finishing with M0407.
- TKLl involve modifications of the endogenous S. cerevisiae loci. In the case of GRE3, both alleles were deleted. For the other four loci, only a single allele was modified. All of the modifications of endogenous loci required the use of selectable antibiotic markers including kan from the Escherichia coR transposon Tn903 (confers resistance to G418), natl from Streptomyces noursei (confers resistance to clonNAT/nourseothricin), and dsdA from Escherichia coli (confers resistance to D-serine.) After selection for a desired genomic modification, the antibiotic marker was excised from the genome using the loxP/cre recombinase system.
- the ere recombinase was carried on plasmid pMU210 which contains a zeocin resistance marker. Loss of pMU210 as well as all antibiotic markers was tested on the appropriate selective media. Subsequent PCR genotyping and DNA sequencing confirmed removal of the antibiotic markers from the modified genomic loci.
- TPI S. cerevisiae triose phosphate isomerase promoter
- M0407 "adapted” colonies were screened in YPDXi media (100 g/L glucose, 50 g/L xylose, 25% MS149 pressate).
- M0407 and M0228 (a xylose-utilizing strain created at Mascoma containing XIyA and XKSl on plasmids) were included as controls. At 24 hours, the glucose had been entirely consumed by all strains. M0407 and M0228 had utilized 30 and 25 g/L of xylose respectively. All nine M0407 "adapted” colonies had utilized more than 44 g/L of xylose. The highest amount of xylose consumed was 48 g/L. This strain was designated M0509.
- 18S rDNA sequencing was used to confirm strain M0509 as Saccharomyces cerevisiae (Kurtzman CP and Robnett, CJ ; FEMS Yeast Research 3 (2003) 417-432).
- a 1774 bp fragment spanning the 18S rDNA was amplified from M0509 genomic DNA and sent for sequencing.
- the 1753 bp of M0509 18S rDNA sequence exhibited a 100% match to the NCBI sequence for S. cerevisiae 18S (nucleotide accession #Z75578).
- strain M0509 was obtained by cultivating M0407 in a chemostat for four weeks, the length of cultivation separating the two strains provides a means to asses the stability of the engineered genetic modifications. Comparision of the DNA sequence of M0407 and M0509 at the GRE3, RKIl, RPEl, TALI, and TKLl loci showed no changes. This suggests that the genetic modifications at these loci are genetically stable, at least under the growth conditions used.
- M0509 was cultivated for -50 generations in liquid media with either glucose or xylose as the sole carbon source. After 50 generations, an individual colony was isolated from each culture and the number of XyIAIXKSl integrations quantified and compared to the original M0509 freezer stock. The colony isolated from the xylose-culture had -20 copies of XyIAIXKSl, the same as the freezer stock. The glucose-cultured colony exhibited a slightly decreased copy number, - 16.
- Example 13 Selection of a Thermotolerant, Robust, Xylose-Utilizing Strain
- Ml 105 is capable of fermentation at temperatures above 40° C in the presence of
- Ml 105 was constructed in a M0509 background and is therefore an industrially robust strain capable of converting both glucose and xylose into ethanol. [0328] Ml 105 was isolated following four rounds of selection / adaptation in a cytostat as outlined in Figure 41 and described as follows. The temperature was increased from 38° C to 41° C during the course of the experiment. Ml 017 (ATCC deposit designation , deposited on November 23, 2009) was isolated from this first cytostat run and was later confirmed by PCR of the GRE3 locus to be a descendant of M0509.
- M1017 was used to inoculate a second cytostat run using YMX media (yeast nitrogen base, 2g/L xylose) at 41° C.
- M1046 was isolated from this second cytostat run.
- M1080 was isolated from a cytostat inoculated with M 1046 and YMX media at 40° C.
- M 1080 grew with a specific growth rate of 0.22 h -1 on YMX at 40° C.
- Ml 105 was isolated from M 1080 based on selection in the cytostat using YPD2X10+acetate media (2 g/L glucose, 10 g/L xylose, 8 g/L acetate, pH 5.4) at 39° C.
- Ml 105 grows 10-20% faster than M0509 in rich media at 35° C.
- Ml 105 has increased acetate tolerance as the strain can grow more quickly than its ancestral strains in the presence of acetate.
- Figure 21 While the parental strains required glucose for tolerance to acetate at high temperatures, Ml 105 does not require glucose or complex medium components to grow in the presence of 7 g/L acetate at pH 5.4.
- Ml 105 was inoculated at approximately 0.7 g/L DCW in 18% MS419 using 3.8 mg Zoomerase/g feedstock at 40° C. Ml 105 produced 3.55% (w/v) ethanol by 168 hours.
- the time course is presented in Figure 22 along with a similar run performed with Ml 088 (described below) for comparison.
- a similar run using only 0.15 g/L DCW for inoculum resulted in 2.9% (w/v) ethanol and some sugar accumulation during the experiment.
- Figure 23 The time course is presented in Figure 22 along with a similar run performed with Ml 088 (described below) for comparison. A similar run using only 0.15 g/L DCW for inoculum resulted in 2.9% (w/v) ethanol and some sugar accumulation during the experiment.
- Example 14 Adaptation of a Thermotolerant, Robust, Xylose-Utilizing Strain
- M1254 is capable of fermentation at temperatures above 40°C in the presence of
- M1254 was isolated following three rounds of selection / adaptation in a cytostat as outlined in Table 11 and Figure 42 and described as follows.
- the first cytostat run was inoculated with Ml 105.
- YMX media yeast nitrogen base w/o amino acids, 20 g/L xylose
- 8g/L acetate was used at pH 5.5 and 40° C.
- Ml 155 was isolated from this first cytostat run and used to inoculate a second cytostat containing YPD media (yeast extract, peptone, 20 g/L glucose) plus 12 g/L acetate at pH 5.4 and 41° C.
- M 1202 was isolated from this second cytostat run.
- M 1254 was isolated from a third cytostat run inoculated with both Ml 155 and M1202 in yeast nitrogen base w/o amino acids + 5% solids equivalent MS419 hydrolysate media at pH 4.8, 39° C. Table 11 : Evolutionary Conditions to Generate M 1254 from Ml 105.
- M1254 grows 7.3 ⁇ 0.9% faster than M1202 and 17 ⁇ 2.0% faster than Ml 155 in 5% solids equivalent MS419 hydrolysate, which is the condition under which strain M 1254 was selected.
- standard fermentation medium limits fermentation performance. Accordingly, use of this strain should be with lower ammonium concentrations, such as 1.1 g/L diammonium phosphate (DAP) or lower than 3 g/L DAP.
- DAP diammonium phosphate
- Figure 24 demonstrates the higher fermentation rate using the lower DAP concentration. The fermentations were performed using 18% MS149, 4 mg external cellulase/g TS, 40° C, 0.5 g/L inoculation DCW M1254 and pH 5.4.
- the pH was controlled using 5 M potassium hydroxide, and 1 g/L magnesium carbonate was fed with each solids feeding. All enzyme was front loaded, while the solids were fed at five time points (0, 3, 6, 24, and 48 hours) in equal size feedings of 3.6% TS.
- MO 1360 was created from M 1254 using the evolutionary conditions described in
- Ml 360 while still substantially inhibited by the synthetic inhibitor mixture, grows at 40° C with a doubling time of approximately 5 hours.
- Figure 25 In industrially relevant medium, Ml 360 is able to generate over 60 g/L ethanol from glucose along with 5 g/L dry cell weight in 48 h at 4O°C beginning with only 60 mg/L dry cell weight.
- Figure 26 In industrially relevant medium, Ml 360 is able to generate over 60 g/L ethanol from glucose along with 5 g/L dry cell weight in 48 h at 4O°C beginning with only 60 mg/L dry cell weight.
- Enzyme activity is known to increase as temperature increases, and thus it is desirable to have thermotolerant S. cerevisiae strains.
- Figure 27 shows three equivalent SSFs with 18% PHW solids loaded. The reactions carried out at 40° C show approximately 17% more ethanol produced than the control reaction carried out at 35° C, when both reactions were carried out at the same external enzyme loading (4mg/g). This increased performance represents a substantial cost savings for the process.
- Example 15 Expression of Cellulases in a Robust Xylose-Utilizing Strain
- Ml 088 is capable of secreting three distinct cellulolytic enzymes: ⁇ -glucosidase from S. fibuligera (SfBGL), cellobiohydrolase 2b from C. lucknowense (ClCBH2b), and cellobiohydrolase I from T. emersonii fused to the T. reesei cellobiohydrolase I cellulose binding domain (TeCBHl+ CBDTrCBHl).
- the M1088 genome also contains genes that encode for polypeptides capable of providing resistance to the following antibiotics: kanamycin, nourseothricin, and hygromycin B.
- Plasmid pMU624 which is also present in M1088, contains a gene encoding for a polypeptide capable of providing resistance to ampicillin.
- the steps used to generate Ml 088 and M0963 from M0509 are summarized in Table 13 below.
- Example 16 Selection of an Endogluconase for Expression in a Robust Xylose- Utilizing Strain
- Endoglucanases augment the activity of cellobiohydrolases, and therefore, the ability of family 5 endoglucanases to complement the previously identified CBHl and CBH2 was invetigated.
- Five family 5 endoglucanses were selected and cloned under control of the ENOl promoter/terminator using the pRDH122 expression plasmid as shown in Table 14.
- Table 14 Family 5 endoglucanases expressed in S. cerevisiae.
- FIG. 28 shows several of the M0749 strains that were spotted on SC -URA plates containing 0.2% of either CMC or lichenin or barley- ⁇ -glucan.
- the M0749 reference strain yielded small zones on the CMC containing plates.
- Both pMU471 (Coptotermes formosanus EG) and pRDH147 based strains yielded very good clearing zones on all the tested substrates.
- the H. jecorina EG2 produced by Y294 and M0749 was visible as ⁇ 57 kDa bands.
- the increased weight compared to the predicted 44 kDA size may represent hyperglycosylation.
- the A. kawachii EGA produced by Y294 was visible as a ⁇ 42 kDa band.
- the A. kawachii EGA produced by M0749 was clearly visible as a ⁇ 120 kDa band.
- the extra weight may signify hyperglycosylation.
- the M0749 strain expressing H.j.eg2 produced the highest levels of secreted activity as measured on PASC or avicel of the EG2s tested. The activity of this enzyme was higher on PASC and avicel than Cf EG (pMU471).
- the synthetic A.kEGA (pRDH145) also gave appreciable activity on both substrates. This product seems to have been produced at higher levels in M0749 than in Y294 and yielded greater activity than CfBG on avicel and PASC when produced in this strain.
- Example 17 Expression of an Endogluconase in Robust Xylose-Utilizing Yeast
- strain M 1403 which contains heterologous genes encoding S. f ⁇ uligera (SfBGL), cellobiohydrolase 2b from C. lucknowense (ClCBH2b), cellobiohydrolase I from T. emersonii fused to the T. reesei cellobiohydrolase I cellulose binding domain (TeCBHl+ CBDTrCBHl), and Heterodera schachtii engl was produced in the M1254 background.
- Strain M1284 which contains heterologous genes encoding those same four cellulases was produced in the M0509 background. Strains M 1284 and M 1403 are described in more detail in Table 15.
- Example 18 Conversion of lignocellulosic substrates via CBP yeast strains
- FIG. 33 presents data from a CBP fermentation of paper sludge by an engineered thermo tolerant S. cerevisiae host strain (parent strain M 1254, cellulo lytic derivative M1403).
- the data for M1254 alone demonstrates that the addition of cellulase ⁇ i.e. zoomerase) is required for ethanol production from paper sludge.
- the data for M 1430 where no external cellulase is added demonstrates that this strain can convert a substantial fraction (-80%) of the "convertible" substrate by virtue of its expressed cellulases. Fermentations with additional external cellulase added to the M 1403 strain demonstrate the ultimate potential of enzymatic conversion for the paper sludge substrate. Visual inspections demonstrated that the non-CBP strain was not able to liquefy the substrate, whereas the CBP strain was.
- BGL can convert paper sludge to a large extent without added cellulase enzyme.
- Figure 34 The control strain in this reaction, M0509, made only a small amount of ethanol during this reaction.
- Ml 179 can convert this material when loaded at lower cell density (1 g/L) as opposed to the higher cell density (10 g/L) used in other reactions. This implies that the strain is able to grow and produce cellulase throughout the fermentation experiments.
- Pretreated hardwood can also be converted by CBP strains.
- Figure 35 shows the effect of using a cellulase expressing strain (M0963), compared to a control strain not expressing cellulases (M0509) during fermentation of PHW.
- M0963 cellulase expressing strain
- M0509 control strain not expressing cellulases
- CBP strains are capable of producing high ethanol titers from PHW as well.
- Figure 36 shows that a 30% washed solids fermentation can generate titers of ethanol up to about 70 g/L with minimal external enzyme loaded 4mg/g and a relatively low cell inoculum (2 g/L). The ability of the low cell density cultivation to eventually catch up to and pass the high cell density culture indicates that the strain grows and continues to make enzyme throughout the fermentation.
- corn stover has been implicated as good substrate for conversion to ethanol via an enzymatic saccharification.
- Figure 37 demonstrates that pretreated corn stover can be converted well by CBP yeast strains.
- the CBP strain in this experiment was able to convert about 82% of what was converted with a high enzyme loading (15 FPU, or about 20 mg/g) could achieve.
- the non-CBP strain made about 60% of the ethanol that the CBP strain was able to achieve.
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EP09828362.5A EP2361311B1 (en) | 2008-11-21 | 2009-11-23 | Yeast expressing cellulases for simultaneous saccharification and fermentation using cellulose |
DK09828362.5T DK2361311T3 (en) | 2008-11-21 | 2009-11-23 | MAKE EXPRESSIVE CELLULASES FOR SIMULTANEOUS INSURANCE AND ACTION BY CELLULOSE |
AU2009316309A AU2009316309B2 (en) | 2008-11-21 | 2009-11-23 | Yeast expressing cellulases for simultaneous saccharification and fermentation using cellulose |
ES09828362.5T ES2621181T3 (en) | 2008-11-21 | 2009-11-23 | Yeasts expressing cellulases for simultaneous saccharification and fermentation using cellulose |
CN2009801537092A CN102272303A (en) | 2008-11-21 | 2009-11-23 | Yeast expressing cellulases for simultaneous saccharification and fermentation using cellulose |
BRPI0921966-8A BRPI0921966B1 (en) | 2008-11-21 | 2009-11-23 | THERMOTOLERANT Yeast HOST CELL, CELLULOSE AND CULTURE HOLDING METHOD |
NZ593469A NZ593469A (en) | 2008-11-21 | 2009-11-23 | Yeast expressing cellulases for simultaneous saccharification and fermentation using cellulose |
US13/130,549 US9102955B2 (en) | 2008-11-21 | 2009-11-23 | Yeast expressing cellulases for simultaneous saccharification and fermentation using cellulose |
CA2744495A CA2744495C (en) | 2008-11-21 | 2009-11-23 | Yeast expressing cellulases for simultaneous saccharification and fermentation using cellulose |
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US15/985,042 US20180258449A1 (en) | 2008-11-21 | 2018-05-21 | Yeast expressing cellulases for simultaneous saccharification and fermentation using cellulose |
US17/131,293 US20210388398A1 (en) | 2008-11-21 | 2020-12-22 | Yeast expressing cellulases for simultaneous saccharification and fermentation using cellulose |
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Family Cites Families (28)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9400623D0 (en) * | 1994-01-14 | 1994-03-09 | Univ Leeds | Exploitation of the cellulase enzyme complex of neurospora |
US7883872B2 (en) | 1996-10-10 | 2011-02-08 | Dyadic International (Usa), Inc. | Construction of highly efficient cellulase compositions for enzymatic hydrolysis of cellulose |
US5811381A (en) | 1996-10-10 | 1998-09-22 | Mark A. Emalfarb | Cellulase compositions and methods of use |
JP3645394B2 (en) | 1997-03-12 | 2005-05-11 | 日立建機株式会社 | Tire roller |
KR100643817B1 (en) * | 2000-06-26 | 2006-11-10 | 유니버시티 오브 플로리다 리서치 파운데이션, 아이엔씨. | Methods and Compositions for Simultaneous Saccharification and Fermentation |
US7045331B2 (en) * | 2001-12-18 | 2006-05-16 | Genencor International, Inc. | EGVII endoglucanase and nucleic acids encoding the same |
US7344876B2 (en) | 2003-01-24 | 2008-03-18 | Phage Biotechnology, Inc. | Kluyveromyces strains metabolizing cellulosic and hemicellulosic materials |
PL1626979T3 (en) * | 2003-05-02 | 2012-09-28 | Cargill Inc | Genetically modified yeast species and fermentation processes using genetically modified yeast |
WO2005116271A2 (en) | 2004-05-25 | 2005-12-08 | The Trustees Of Dartmouth College | Selection of microorganisms with growth dependent upon extracytoplasmic enzymes |
WO2006009434A1 (en) * | 2004-07-16 | 2006-01-26 | Technische Universiteit Delft | Metabolic engineering of xylose fermenting eukaryotic cells |
WO2008008793A2 (en) | 2006-07-10 | 2008-01-17 | Dyadic International Inc. | Methods and compositions for degradation of lignocellulosic material |
CA2670102A1 (en) * | 2006-11-22 | 2008-05-29 | The Trustees Of Dartmouth College | Recombinant yeast strains expressing tethered cellulase enzymes |
WO2008155665A2 (en) | 2007-05-09 | 2008-12-24 | University Of Stellenbosch | Method for enhancing cellobiose utilization |
US7923236B2 (en) | 2007-08-02 | 2011-04-12 | Dyadic International (Usa), Inc. | Fungal enzymes |
WO2009138877A2 (en) | 2008-05-11 | 2009-11-19 | Universiteit Stellenbosch | Heterologous expression of fungal cellobiohydrolases in yeast |
US9994835B2 (en) | 2008-05-11 | 2018-06-12 | Lallemand Hungary Liquidity Management Llc | Construction of protrophic/celluloytic yeast strains expressing tethered and secreted cellulases |
WO2010005551A2 (en) | 2008-07-07 | 2010-01-14 | Mascoma Corporation | Heterologous expression of termite cellulases in yeast |
WO2010005553A1 (en) | 2008-07-07 | 2010-01-14 | Mascoma Corporation | Isolation and characterization of schizochytrium aggregatum cellobiohydrolase i (cbh 1) |
AU2009316309B2 (en) * | 2008-11-21 | 2016-07-07 | Lallemand Hungary Liquidity Management Llc | Yeast expressing cellulases for simultaneous saccharification and fermentation using cellulose |
US9206434B2 (en) | 2008-12-23 | 2015-12-08 | Enchi Corporation | Heterologous biomass degrading enzyme expression in thermoanaerobacterium saccharolyticum |
US9315833B2 (en) | 2009-02-20 | 2016-04-19 | Lallemand Hungary Liquidity Management Llc | Yeast cells expressing an exogenous cellulosome and methods of using the same |
WO2010124000A2 (en) | 2009-04-21 | 2010-10-28 | The Trustees Of Dartmouth College | Modified cipa gene from clostridium thermocellum for enhanced genetic stability |
BR112012003883A8 (en) | 2009-08-21 | 2018-02-06 | Mascoma Corp | RECOMBINANT MICROORGANISMS, PROCESS OF CONVERSION OF LIGNOCELLULOSIC BIOMASS INTO 1,2-PROPANEDIOL OR ISOPROPANOL, ENGINEERED METABOLIC VIA, GENETIC Clustering, AND METHOD OF IDENTIFICATION OF VITAMIN B12 INDEPENDENT DIOL DEHYDRATASE THAT CONVERTS PROPANEDIOL TO PROPANAL |
BR112012028290B1 (en) | 2010-05-05 | 2021-02-02 | Lallemand Hungary Liquidity Management Llc. | recombinant yeast, process to convert biomass into ethanol and fermentation medium comprising said yeast |
CN103124783A (en) | 2010-06-03 | 2013-05-29 | 马斯科马公司 | Yeast expressing saccharolytic enzymes for consolidated bioprocessing using starch and cellulose |
FI20105632A0 (en) * | 2010-06-04 | 2010-06-04 | Valtion Teknillinen | Procedure for protein production in filamentous fungi |
US9745560B2 (en) | 2012-08-29 | 2017-08-29 | Lallemand Hungary Liquidity Management Llc | Expression of enzymes in yeast for lignocellulose derived oligomer CBP |
EP2970861A2 (en) | 2013-03-15 | 2016-01-20 | Lallemand Hungary Liquidity Management LLC | Expression of beta-glucosidases for hydrolysis of lignocellulose and associated oligomers |
-
2009
- 2009-11-23 AU AU2009316309A patent/AU2009316309B2/en not_active Ceased
- 2009-11-23 EP EP09828362.5A patent/EP2361311B1/en active Active
- 2009-11-23 WO PCT/US2009/065571 patent/WO2010060056A2/en active Application Filing
- 2009-11-23 CA CA2744495A patent/CA2744495C/en active Active
- 2009-11-23 US US13/130,549 patent/US9102955B2/en active Active
- 2009-11-23 DK DK09828362.5T patent/DK2361311T3/en active
- 2009-11-23 CN CN2009801537092A patent/CN102272303A/en active Pending
- 2009-11-23 NZ NZ593469A patent/NZ593469A/en unknown
- 2009-11-23 ES ES09828362.5T patent/ES2621181T3/en active Active
- 2009-11-23 CN CN201710766848.9A patent/CN108315272A/en active Pending
- 2009-11-23 CA CA2964245A patent/CA2964245C/en active Active
- 2009-11-23 BR BRPI0921966-8A patent/BRPI0921966B1/en active IP Right Grant
- 2009-11-23 CA CA3092340A patent/CA3092340A1/en active Pending
-
2015
- 2015-07-01 US US14/788,879 patent/US9988652B2/en active Active
-
2018
- 2018-05-21 US US15/985,042 patent/US20180258449A1/en not_active Abandoned
-
2020
- 2020-12-22 US US17/131,293 patent/US20210388398A1/en active Pending
Non-Patent Citations (4)
Title |
---|
MCBRIDE, J.E. ET AL., ENZYME MICROB. TECHOL., vol. 37, 2005, pages 93 - 101 |
See also references of EP2361311A4 |
VAN RENSBURG, P. ET AL., YEAST, vol. 14, 1998, pages 67 - 76 |
VAN ROOYEN, R. ET AL., J. BIOTECH., vol. 120, 2005, pages 284 - 295 |
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US9598700B2 (en) | 2010-06-25 | 2017-03-21 | Agrivida, Inc. | Methods and compositions for processing biomass with elevated levels of starch |
US10443068B2 (en) | 2010-06-25 | 2019-10-15 | Agrivida, Inc. | Plants with engineered endogenous genes |
CN103328642B (en) * | 2010-12-22 | 2016-03-09 | 耐思特石油公司 | For the production of the integrated approach of biofuel |
CN103328642A (en) * | 2010-12-22 | 2013-09-25 | 耐思特石油公司 | An integrated process for producing biofuels |
JP2014500036A (en) * | 2010-12-22 | 2014-01-09 | ネステ オイル オサケ ユキチュア ユルキネン | Integrated method for producing biofuels |
WO2012085340A1 (en) * | 2010-12-22 | 2012-06-28 | Neste Oil Oyj | An integrated process for producing biofuels |
US9434962B2 (en) | 2010-12-22 | 2016-09-06 | Neste Oyj | Integrated process for producing biofuels |
AU2011347057B2 (en) * | 2010-12-22 | 2015-12-17 | Neste Oil Oyj | An integrated process for producing biofuels |
US9353363B2 (en) | 2011-01-26 | 2016-05-31 | Novozymes A/S | Glycoside hydrolases from thermophilic fungi |
US10647971B2 (en) | 2011-01-26 | 2020-05-12 | Novozymes A/S | Glycoside hydrolases from thermophlic fungi |
US10626385B2 (en) | 2011-01-26 | 2020-04-21 | Novozymes A/S | Polypeptides having cellobiohydrolase activity and polynucleotides encoding same |
US10233435B2 (en) | 2011-01-26 | 2019-03-19 | Novozymes A/S | Glycoside hydrolases from thermophlic fungi |
US9506048B2 (en) | 2011-01-26 | 2016-11-29 | Novozymes, Inc. | Polypeptides having cellobiohydrolase activity and polynucleotides encoding same |
WO2012122308A3 (en) * | 2011-03-07 | 2012-11-08 | Agrivida, Inc. | Consolidated pretreatment and hydrolysis of plant biomass expressing cell wall degrading enzymes |
US11034967B2 (en) | 2011-04-05 | 2021-06-15 | Lallemand Hungary Liquidity Management Llc | Methods for the improvement of product yield and production in a microorganism through the addition of alternate electron acceptors |
US8956851B2 (en) | 2011-04-05 | 2015-02-17 | Lallemand Hungary Liquidity Management, LLC | Methods for the improvement of product yield and production in a microorganism through the addition of alternate electron acceptors |
US9719098B2 (en) | 2011-04-05 | 2017-08-01 | Lallemand Hungary Liquidity Management Llc | Methods for the improvement of product yield and production in a microorganism through the addition of alternate electron acceptors |
US9598689B2 (en) | 2011-11-10 | 2017-03-21 | Lallemand Hungary Liquidity Management Llc | Genetically modified strain of S. cerevisiae engineered to ferment xylose and arabinose |
US10047380B2 (en) | 2011-11-10 | 2018-08-14 | Lallemand Hungary Liquidity Management Llc | Genetically modified strain of S. cerevisiae engineered to ferment xylose and arabinose |
US10059933B2 (en) | 2011-11-18 | 2018-08-28 | Novozymes Inc. | Polypeptides having endoglucanase activity and polynucleotides encoding same |
US9879242B2 (en) | 2011-11-18 | 2018-01-30 | Novozymes Inc. | Polypeptides having endoglucanase activity and polynucleotides encoding same |
US9708591B2 (en) | 2011-11-18 | 2017-07-18 | Novozymes Inc. | Polypeptides having endoglucanase activity and polynucleotides encoding same |
WO2013090053A1 (en) * | 2011-12-13 | 2013-06-20 | Danisco Us Inc. | Enzyme cocktails prepared from mixed cultures |
CN104039834B (en) * | 2011-12-22 | 2018-09-14 | 诺维信股份有限公司 | Hybrid polypeptide with cellobiohydrolase activity and their polynucleotides of coding |
US9481897B2 (en) | 2011-12-22 | 2016-11-01 | Novozymes, Inc. | Hybrid polypeptides having cellobiohydrolase activity and polynucleotides encoding same |
US9969994B2 (en) | 2011-12-22 | 2018-05-15 | Novozymes, Inc. | Hybrid polypeptides having cellobiohydrolase activity and polynucleotides encoding same |
CN104039834A (en) * | 2011-12-22 | 2014-09-10 | 诺维信股份有限公司 | Hybrid polypeptides having cellobiohydrolase activity and polynucleotides encoding same |
EP2794664A4 (en) * | 2011-12-22 | 2015-08-19 | Novozymes Inc | Hybrid polypeptides having cellobiohydrolase activity and polynucleotides encoding same |
US20140356925A1 (en) * | 2012-02-17 | 2014-12-04 | Kabushiki Kaisha Toyota Chuo Kenkyusho | Polypeptide capable of enhancing cellulosic biomass degradation |
US20150252340A1 (en) * | 2012-10-31 | 2015-09-10 | Danisco Us Inc. | Compositions and methods of us |
US20150252343A1 (en) * | 2012-10-31 | 2015-09-10 | Danisco Us Inc. | Beta-glucosidase from magnaporthe grisea |
WO2014074895A2 (en) | 2012-11-09 | 2014-05-15 | Mascoma Corporation | Method for acetate consumption during ethanolic fermentation of cellulosic feedstocks |
EP3498829A1 (en) | 2012-11-09 | 2019-06-19 | Lallemand Hungary Liquidity Management LLC | Method for acetate consumption during ethanolic fermentation of cellulosic feedstocks |
CN105229141A (en) * | 2013-03-15 | 2016-01-06 | 拉勒曼德匈牙利流动管理有限责任公司 | For the expression of the beta-glucosidase enzyme of the hydrolysis of lignocellulose and relevant oligopolymer |
WO2014151805A3 (en) * | 2013-03-15 | 2015-01-08 | Lallemand Hungary Liquidity Management Llc | Expression of beta-glucosidases for hydrolysis of lignocellulose and associated oligomers |
US10612061B2 (en) | 2013-03-15 | 2020-04-07 | Lallemand Hungary Liquidity Management Llc | Expression of beta-glucosidases for hydrolysis of lignocellulose and associated oligomers |
WO2014151805A2 (en) | 2013-03-15 | 2014-09-25 | Mascoma Corporation | Expression of beta-glucosidases for hydrolysis of lignocellulose and associated oligomers |
US11168315B2 (en) | 2013-03-15 | 2021-11-09 | Lallemand Hungary Liquidity Management Llc | Expression of beta-glucosidases for hydrolysis of lignocellulose and associated oligomers |
CN103409333A (en) * | 2013-05-20 | 2013-11-27 | 山东大学 | Recombinant saccharomyces cerevisiae strain for continuously and efficiently secreting beta-glucosidase and applications thereof |
US11753656B2 (en) | 2013-08-15 | 2023-09-12 | Lallemand Hungary Liquidity Management Llc | Methods for the improvement of product yield and production in a microorganism through glycerol recycling |
WO2015180362A1 (en) * | 2014-05-29 | 2015-12-03 | 中国科学院广州能源研究所 | Mesophile ethanol-tolerant beta-glucosidase and coding gene and use thereof |
US11390898B2 (en) | 2014-09-05 | 2022-07-19 | Novozymes A/S | Polypeptides having cellobiohydrolase activity and polynucleotides encoding same |
EP3594335A1 (en) | 2014-09-05 | 2020-01-15 | Novozymes A/S | Carbohydrate binding module variants and polynucleotides encoding same |
WO2016037096A1 (en) | 2014-09-05 | 2016-03-10 | Novozymes A/S | Carbohydrate binding module variants and polynucleotides encoding same |
US10676769B2 (en) | 2014-09-08 | 2020-06-09 | Novozymes A/S | Cellobiohydrolase variants and polynucleotides encoding same |
CN104357340A (en) * | 2014-11-17 | 2015-02-18 | 中国食品发酵工业研究院 | Method for producing L-malic acid by virtue of biotransformation of fumaric acid |
EP3739045A2 (en) | 2015-02-24 | 2020-11-18 | Novozymes A/S | Cellobiohydrolase variants and polynucleotides encoding same |
WO2016138167A2 (en) | 2015-02-24 | 2016-09-01 | Novozymes A/S | Cellobiohydrolase variants and polynucleotides encoding same |
US10557127B2 (en) | 2015-02-24 | 2020-02-11 | Novozymes A/S | Cellobiohydrolase variants and polynucleotides encoding same |
WO2018215956A1 (en) | 2017-05-23 | 2018-11-29 | Lallemand Hungary Liquidity Management Llc. | Optimization of biomass-based fermentations |
US11286508B2 (en) * | 2017-12-19 | 2022-03-29 | Universidad Del País Vasco | Ancestral cellulases and uses thereof |
CN108823102A (en) * | 2018-06-10 | 2018-11-16 | 东北农业大学 | Cold ground straw decomposing fungi Mortierella bacterial strain and its application in rice straw is decomposed |
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US9988652B2 (en) | 2018-06-05 |
US20120129229A1 (en) | 2012-05-24 |
US9102955B2 (en) | 2015-08-11 |
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CA3092340A1 (en) | 2010-05-27 |
CN102272303A (en) | 2011-12-07 |
CA2964245C (en) | 2020-10-27 |
NZ593469A (en) | 2012-12-21 |
CA2744495C (en) | 2017-05-30 |
EP2361311A4 (en) | 2013-03-27 |
CN108315272A (en) | 2018-07-24 |
CA2964245A1 (en) | 2010-05-27 |
EP2361311A2 (en) | 2011-08-31 |
BRPI0921966A2 (en) | 2017-10-24 |
CA2744495A1 (en) | 2010-05-27 |
AU2009316309A1 (en) | 2011-10-20 |
US20210388398A1 (en) | 2021-12-16 |
BRPI0921966B1 (en) | 2019-05-07 |
AU2009316309B2 (en) | 2016-07-07 |
US20180258449A1 (en) | 2018-09-13 |
US20160010117A1 (en) | 2016-01-14 |
BRPI0921966A8 (en) | 2017-12-12 |
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