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Compositions and methods for production of fermentable sugars

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20120270278 patent thumbnailZoom

Compositions and methods for production of fermentable sugars


The present application provides genetically modified fungal organisms that produce enzyme mixtures exhibiting enhanced hydrolysis of cellulosic material to glucose, enzyme mixtures produced by the genetically modified fungal organisms, and processes for producing glucose from cellulose using such enzyme mixtures.

Browse recent Codexis, Inc. patents - Redwood City, CA, US
Inventors: ISH KUMAR DHAWAN, DIPNATH BAIDYAROY, ANDREW SHAW, OLEH TANCHAK, CHRISTOPHER HILL, CHENGSONG LIU, AMALA CHOKSHI, BRIAN R. SCOTT
USPTO Applicaton #: #20120270278 - Class: 435100 (USPTO) - 10/25/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition >Preparing Compound Containing Saccharide Radical >Disaccharide

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The Patent Description & Claims data below is from USPTO Patent Application 20120270278, Compositions and methods for production of fermentable sugars.

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The present application is a Divisional of U.S. patent application Ser. No. 13/286,972, filed Nov. 1, 2011, which claims priority to U.S. Prov. Patent Appln. Ser. Nos. 61/409,186, 61/409,217, 61/409,472, and 61/409,480, all of which were filed on Nov. 2, 2010, and are hereby incorporated by reference herein.

FIELD OF THE INVENTION

The present invention provides compositions and methods for the production of fermentable sugars. In some embodiments, the present invention provides genetically modified fungal organisms. In some additional embodiments, the present invention provides enzymes that find use in enhancing hydrolysis of cellulosic material to fermtable sugars (e.g., glucose), and methods for using the enzymes. In some further embodiments, the present invention provides enzyme mixtures useful for the hydrolysis of cellulosic materials.

BACKGROUND

Cellulose is a polymer of the simple sugar glucose linked by beta-1,4 glycosidic bonds. Many microorganisms produce enzymes that hydrolyze beta-linked glucans. These enzymes include endoglucanases, cellobiohydrolases, and beta-glucosidases. Endoglucanases digest the cellulose polymer at random locations, opening it to attack by cellobiohydrolases. Cellobiohydrolases sequentially release molecules of cellobiose from the ends of the cellulose polymer. Cellobiose is a water-soluble beta-1,4-linked dimer of glucose. Beta-glucosidases hydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has the advantages of the ready availability of large amounts of feedstock, the desirability of avoiding burning or land filling the materials, and lower overall greenhouse gas production. Wood, agricultural residues, herbaceous crops, and municipal solid wastes have been considered as feedstocks for ethanol production. These materials primarily consist of cellulose, hemicellulose, and lignin. Once the cellulose is converted to glucose, the glucose is easily fermented by yeast into ethanol.

Although progress has been made in increasing the efficiency of enzymatic degradation of lignocellulosic feedstocks, there remains a great need to improve yield of fermentable sugars using enzymatic processes.

SUMMARY

OF THE INVENTION

The present invention provides genetically modified fungal organisms, as well as enzymes that enhance hydrolysis of cellulosic material to glucose, and methods for using the enzymes.

The present invention provides fungal cells that have been genetically modified to reduce the amount of endogenous glucose and/or cellobiose oxidizing enzyme activity that is produced by the fungal cells. In some embodiments, the fungal cell is an Ascomycete belonging to the subdivision Pezizomycotina, and/or wherein the fungal cell is from the family Chaetomiaceae. In some embodiments, the fungal cell is a species of Myceliophthora, Thielavia, Sporotrichum, Neurospora, Sordaria, Podospora, Magnaporthe, Fusarium, Gibberella, Botryotinia, Humicola, Neosartorya, Pyrenophora, Phaeosphaeria, Sclerotinia, Chaetomium, Nectria, Verticillium, Corynascus, Acremonium, Ctenomyces, Chrysosporium, Scytalidium, Talaromyces, Thermoascus, or Aspergillus. In some additional embodiments, the fungal cell is a species of Myceliophthora, Thielavia, Sporotrichum, Chrysosporium, Corynascus, Acremonium, Chaetomium, Ctenomyces, Scytalidium, Talaromyces, or Thermoascus, while in some other embodiments, the fungal cell is Sporotrichum thermophile Sporotrichum cellulophilum, Thielavia heterothallica, Thielavia terrestris, Corynascus heterothallicus, or Myceliophthora thermophile. In some embodiments, the fungal cell has been genetically modified to reduce the amount of endogenous glucose oxidase and/or cellobiose dehydrogenase that is produced by the fungal cell. In some additional embodiments, the fungal cell has been genetically modified to reduce the amount of endogenous glucose oxidase and/or cellobiose dehydrogenase that is produced by the fungal cell and to increase the production of at least one saccharide hydrolyzing enzyme. In some further embodiments, the fungal cell has been genetically modified to reduce the amount of endogenous glucose oxidase and/or cellobiose dehydrogenase that is produced by the fungal cell and to increase the production of at least one saccharide hydrolyzing enzyme, and wherein the fungal cell is a Basidiomycete belonging to the class Agaricomycetes. In some embodiments, the Basidiomycete is a species of Pleurotus, Peniophora, Trametes, Athelia, Sclerotium, Termitomyces, Flammulina, Coniphora, Ganoderma, Pycnoporus, Ceriporiopsis, Phanerochaete, Gloeophyllum, Hericium, Heterobasidion, Gelatoporia, Lepiota, or Irpex. In some embodiments, the fungal cell has been genetically modified to reduce the amount of the endogenous glucose oxidase and/or cellobiose dehydrogenase that is secreted by the fungal cell. In some additional embodiments, the fungal cell has been genetically modified to disrupt the secretion signal peptide of the glucose and/or cellobiose oxidizing enzyme. In some further embodiments, the fungal cell has been genetically modified to reduce the amount of the endogenous glucose and/or cellobiose oxidizing enzyme that is expressed by the fungal cell. In still some additional embodiments, the fungal cell has been genetically modified to disrupt a translation initiation sequence in the transcript encoding the endogenous glucose and/or cellobiose oxidizing enzyme. In some additional embodiments, the fungal cell has been genetically modified to introduce a frameshift mutation in the transcript encoding the endogenous glucose and/or cellobiose oxidizing enzyme. In some further embodiments, the fungal cell has been genetically modified to reduce the transcription level of a gene encoding the endogenous glucose and/or cellobiose oxidizing enzyme. In some embodiments, the fungal cell has been genetically modified to disrupt the promoter of a gene encoding the endogenous glucose and/or cellobiose oxidizing enzyme. In still some additional embodiments, the fungal cell has been genetically modified to at least partially delete at least one gene encoding the endogenous glucose and/or cellobiose oxidizing enzyme. In some further embodiments, the fungal cell has been genetically modified to reduce the catalytic efficiency of the endogenous glucose and/or cellobiose oxidizing enzyme. In some additional embodiments, the fungal cell has been genetically modified to mutate one or more residues in an active site of the glucose and/or cellobiose oxidizing enzyme. In some further embodiments, the fungal cell has been genetically modified to mutate one or more residues in a heme binding domain of the glucose and/or cellobiose oxidizing enzyme. In some embodiments of the fungal cells provided herein, the glucose and/or cellobiose oxidizing enzyme is selected from cellobiose dehydrogenase (EC 1.1.99.18), glucose oxidase (EC 1.1.3.4), pyranose oxidase (EC1.1.3.10), glucooligosaccharide oxidase (EC 1.1.99.B3), pyranose dehydrogenase (EC 1.1.99.29), and glucose dehydrogenase (EC 1.1.99.10). In some additional embodiments, the glucose and/or cellobiose oxidizing enzyme comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, and/or 16. In some further embodiments, the glucose and/or cellobiose oxidizing enzyme is cellobiose dehydrogenase (EC 1.1.99.18). In some embodiments, the fungal cell has been genetically modified to reduce the amount of glucose and/or cellobiose oxidizing enzyme activity of two or more endogenous glucose and/or cellobiose oxidizing enzymes that are produced by the fungal cell prior to genetic modification. In some further embodiments, the first of the two or more the glucose and/or cellobiose oxidizing enzymes comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, and/or 16, and a second of the two or more the glucose and/or cellobiose oxidizing enzymes comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, and/or 16.

The present invention also provides enzyme mixtures comprising two or more cellulose hydrolyzing enzymes, wherein at least one of the two or more cellulose hydrolyzing enzymes is expressed by at least one of the fungal cells provided herein.

The present invention also provides enzyme mixtures comprising two or more cellulose hydrolyzing enzymes, wherein at least one of the two or more cellulose hydrolyzing enzymes is produced by a fungal cell that has been genetically modified to reduce the amount of endogenous glucose and/or cellobiose oxidizing enzyme activity that is secreted by the fungal cell, and wherein the fungal cell is an Ascomycete belonging to the subdivision Pezizomycotina. In some embodiments, the fungal cell is a species of Myceliophthora, Thielavia, Sporotrichum, Corynascus, Acremonium, Chaetomium, Ctenomyces, Scytalidium, Talaromyces, or Thermoascus.

The present invention also provides enzyme mixtures comprising two or more cellulose hydrolyzing enzymes, wherein at least one of the two or more cellulose hydrolyzing enzymes is produced by a fungal cell that has been genetically modified to reduce the amount of endogenous glucose and/or cellobiose oxidizing enzyme activity that is secreted by the fungal cell and to increase the production of at least one saccharide hydrolyzing enzyme, wherein the fungal cell is a Basidiomycete belonging to the class Agaricomycetes.

In some embodiments, the enzyme mixtures are cell-free mixtures. In some additional embodiments, a substrate of the enzyme mixture comprises pretreated lignocellulose. In some further embodiments, the pretreated lignocellulose comprises lignocellulose treated by a treatment method selected from acid pretreatment, ammonium pretreatment, steam explosion and/or organic solvent extraction.

The present invention also provides enzyme mixtures comprising two or more cellulose hydrolyzing enzymes, wherein the fungal cellulase enzyme mixture is modified relative to a parental (or reference) enzyme mixture to be at least partially deficient in glucose and/or cellobiose oxidizing enzyme activity.

The present invention further provides enzyme mixtures comprising two or more cellulose hydrolyzing enzymes, at least one of the cellulose hydrolyzing enzymes being endogenous to a fungal cell, wherein the fungal cell is a Basidiomycete belonging to the class Agaricomycetes or an Ascomycete belonging to the subdivision Pezizomycotina and wherein the enzyme mixture is characterized in that, when the enzyme mixture is contacted with cellobiose and/or glucose, no more than about 10%, about 15% or about 20%, of the cellobiose and/or glucose is oxidized after 10 hours.

In some embodiments of the enzyme mixtures, the fungal cell has been genetically modified to reduce the amount of glucose and/or cellobiose oxidase enzyme activity that is secreted by the fungal cell. In some further embodiments, the enzyme mixture is a cell-free mixture. In some additional embodiments, the enzyme mixture comprises at least one beta-glucosidase. In some further embodiments, the enzyme mixture comprises at least one cellulase enzyme selected from endoglucanases (EGs), beta-glucosidases (BGLs), Type 1 cellobiohydrolases (CBH1s), Type 2 cellobiohydrolases (CBH2s), and/or glycoside hydrolase 61s (GH61s), and/or variants of the cellulase enzyme. In some embodiments, the enzyme mixture further comprises at least one cellobiose dehydrogenase. In some embodiments, the celliobiose dehydrogenase is CDH1 and/or CDH2. In some additional embodiments, the enzyme mixture further comprises at least one cellulase enzyme and/or at least one additional enzyme. In some further embodiments, the enzyme mixture has been subjected to a purification process to selectively remove one or more glucose and/or cellobiose oxidizing enzymes from the enzyme mixture. In some embodiments, the purification process comprises selective precipitation to separate the glucose and/or cellobiose oxidizing enzymes from other enzymes present in the enzyme mixture. In some additional embodiments, the enzyme mixtures comprise at least one inhibitor of one or more glucose and/or cellobiose oxidizing enzymes.

The present invention also provides methods for generating cellobiose and/or glucose comprising contacting a cellulose substrate with an enzyme mixture comprising two or more cellulose hydrolyzing enzymes to generate glucose and/or cellobiose, wherein at least one of the cellulose hydrolyzing enzymes is endogenous to a fungal cell that is an Ascomycete belonging to the subdivision Pezizomycotina, and wherein the enzyme mixture is characterized in that, when the enzyme mixture is contacted with cellobiose and/or glucose, no more than about 10%, about 15%, or about 20% of the cellobiose and/or glucose is oxidized after 10 hours. In some embodiments, the Ascomycete is a species of Myceliophthora, Thielavia, Sporotrichum, Neurospora, Sordaria, Podospora, Magnaporthe, Fusarium, Gibberella, Botryotinia, Humicola, Neosartorya, Pyrenophora, Phaeosphaeria, Sclerotinia, Chaetomium, Nectria, Verticillium, or Aspergillus.

The present invention also provides methods for generating cellobiose and/or glucose comprising contacting a cellulose substrate with an enzyme mixture comprising two or more cellulose hydrolyzing enzymes to generate glucose and/or cellobiose, wherein at least one of the cellulose hydrolyzing enzymes is endogenous to a fungal cell that is a Basidiomycete belonging to the class Agaricomycetes, and wherein the enzyme mixture is characterized in that, when the enzyme mixture is contacted with cellobiose and/or glucose, no more than about 10%, about 15% or about 20% of the cellobiose and/or glucose is oxidized after 10 hours. In some embodiments, the Basidiomycete is a species of Pleurotus, Peniophora, Trametes, Athelia, Sclerotium, Termitomyces, Flammulina, Coniphora, Ganoderma, Pycnoporus, Ceriporiopsis, Phanerochaete, Gloeophyllum, Hericium, Heterobasidion, Gelatoporia, Lepiota, or Irpex.

The present invention also provides methods for generating cellobiose and/or glucose comprising contacting a cellulose substrate with an enzyme mixture comprising two or more cellulose hydrolyzing enzymes to generate glucose and/or cellobiose, wherein at least one of the cellulose hydrolyzing enzymes is endogenous to a fungal cell that is an Ascomycete belonging to the subdivision Pezizomycotina, and wherein, of the cellulose hydrolyzed by the enzyme mixture, at least about 80%, about 85%, or about 90% is present in the form of cellobiose and/or glucose. In some embodiments, the Ascomycete is a species of Myceliophthora, Thielavia, Sporotrichum, Neurospora, Sordaria, Podospora, Magnaporthe, Fusarium, Gibberella, Botryotinia, Humicola, Neosartorya, Pyrenophora, Phaeosphaeria, Sclerotinia, Chaetomium, Nectria, Verticillium, or Aspergillus. In some embodiments, the Ascomycete is Myceliophthora thermophila, Thielavia heterothallica or Sporotrichum thermophile. In some embodiments, the fungal cell is Myceliophthora thermophila.

The present invention also provides methods for generating cellobiose and/or glucose comprising contacting a cellulose substrate with an enzyme mixture comprising two or more cellulose hydrolyzing enzymes to generate glucose and/or cellobiose, wherein at least one of the cellulose hydrolyzing enzymes is endogenous to a fungal cell that is a Basidiomycete belonging to the class Agaricomycetes, and wherein, of the cellulose hydrolyzed by the enzyme mixture, at least about 80%, about 85%, or about 90% is present in the form of cellobiose and/or glucose. In some embodiments, the Basidiomycete is a species of Pleurotus, Peniophora, Trametes, Athelia, Sclerotium, Termitomyces, Flammulina, Coniphora, Ganoderma, Pycnoporus, Ceriporiopsis, Phanerochaete, Gloeophyllum, Hericium, Heterobasidion, Gelatoporia, Lepiota, or Irpex.

The present invention also provides methods for producing cellobiose and/or glucose from cellulose comprising treating a cellulose substrate with an enzyme mixture to generate glucose, wherein the enzyme mixture is modified relative to a secreted enzyme mixture from a reference (or parental) fungal cell to be at least partially deficient in glucose and/or cellobiose oxidizing enzyme activity. In some embodiments of the methods, the enzyme mixture is a cell-free mixture. In some additional embodiments, the cellulose substrate comprises pretreated lignocellulose. In some further embodiments, the pretreated lignocellulose comprises lignocellulose treated by a treatment method selected from acid pretreatment, ammonium pretreatment, steam explosion and/or organic solvent extraction. In some further embodiments, the methods further comprise fermentation of the cellobiose and/or glucose to at least one end product. In some embodiments, the end product is at least one fuel alcohol and/or at least one precursor industrial chemical. In some additional embodiments, the fuel alcohol is ethanol or butanol. In some embodiments, the process for producing cellobiose and/or glucose from cellulose and said fermentation are conducted in a simultaneous saccharification and fermentation (SSF) process. In some further additional embodiments, the enzyme mixture is produced by a fungal cell has that been genetically modified to reduce the amount of one or more endogenous glucose and/or cellobiose oxidizing enzymes that is secreted by the fungal cell. In some embodiments, the enzyme mixture has been subjected to a purification process to selectively remove at least one glucose and/or cellobiose oxidizing enzyme from the enzyme mixture. In some further embodiments, the purification process comprises selective precipitation to separate the glucose and/or cellobiose oxidizing enzyme from other enzymes present in the enzyme mixture. In still some additional embodiments, the enzyme mixture comprises at least one inhibitor of the glucose and/or cellobiose oxidizing enzyme. In some embodiments, the inhibitor comprises a broad-spectrum oxidase inhibitor selected from sodium azide, potassium cyanide, a metal anion, and a combination thereof. In some embodiments, the inhibitor comprises a specific inhibitor of cellobiose dehydrogenase (EC 1.1.99.18) selected from cellobioimidazole, gentiobiose, lactobiono-1,5-lactone, celliobono-1,5-lactone, tri-N-acetylchitortriose, methyl-beta-D cellobiosidase, 2,2-bipyridine, cytochrome C, and a combination thereof. In some embodiments, the method is a batch process, while in some other embodiments it is a continuous process, and in some further embodiments it is a fed-batch process and in still further embodiments, it is a combination of batch, continuous and/or fed-batch processes conducted in any order. In some embodiments, the method is conducted in a reaction volume of at least 10,000 liters, while in some other embodiments, the method is conducted in a reaction volume of at least 100,000 liters. In some embodiments, the enzyme mixture comprises at least one beta-glucosidase, while in some other embodiments, the enzyme mixture does not comprise a beta-glucosidase. In some embodiments, the enzyme mixture comprises at least one endoglucanase, while in some other embodiments, the enzyme mixture does not comprise an endoglucanase. In some embodiments, the enzyme mixture comprises at least one cellulase enzyme selected from endoglucanases (EGs), beta-glucosidases (BGLs), Type 1 cellobiohydrolases (CBH1s), Type 2 cellobiohydrolases (CBH2s), and/or glycoside hydrolase 61s (GH61s), and/or variants of said cellulase enzyme.

The present invention also provides methods for generating glucose comprising contacting cellulose with an enzyme mixture comprising two or more cellulose hydrolyzing enzymes, wherein at least one of the two or more cellulose hydrolyzing enzymes is produced by the fungal cells provided herein.

The present invention also provides methods for generating glucose comprising contacting cellulose with an enzyme mixture comprising two or more cellulose hydrolyzing enzymes, wherein at least one of the two or more cellulose hydrolyzing enzymes is produced by a fungal cell that has been genetically modified to reduce the amount of endogenous glucose and/or cellobiose oxidizing enzyme activity that is secreted by the fungal cell, wherein the fungal cell is an Ascomycete belonging to the subdivision Pezizomycotina. In some embodiments, the fungal cell is a species of Myceliophthora, Thielavia, Sporotrichum, Corynascus, Acremonium, Chaetomium, Ctenomyces, Scytalidium, Talaromyces, or Thermoascus.

The present invention also provides methods for generating glucose comprising contacting cellulose with an enzyme mixture comprising two or more cellulose hydrolyzing enzymes, wherein at least one of the two or more cellulose hydrolyzing enzymes is produced by a fungal cell that has been genetically modified to reduce the amount of endogenous glucose and/or cellobiose oxidizing enzyme activity that is secreted by the fungal cell and to increase the production of at least one saccharide hydrolyzing enzyme, wherein the fungal cell is a Basidiomycete belonging to the class Agaricomycetes.

The present invention further provides methods for generating glucose comprising contacting cellulose with at least one enzyme mixture as provided herein. In some embodiments, the cellulose comprises pretreated lignocellulose. In some additional embodiments, the pretreated lignocellulose comprises lignocellulose treated by a treatment method selected from acid pretreatment, ammonium pretreatment, steam explosion and/or organic solvent extraction. In some additional embodiments, the enzyme mixture is a cell-free mixture. In some further embodiments, the methods further comprise fermentation of the glucose to an end product. In some embodiments, the end product is a fuel alcohol or a precursor industrial chemical. In some embodiments, the fuel alcohol is ethanol or butanol.



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stats Patent Info
Application #
US 20120270278 A1
Publish Date
10/25/2012
Document #
13539799
File Date
07/02/2012
USPTO Class
435100
Other USPTO Classes
435 72, 435155, 435160, 435165
International Class
/
Drawings
14



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