Iogen Energy Corporation

Iogen Corporation

Iogen Bio-Products Corporation

Her Majesty the Queen in Right of Canada, as Represented by the Minister

Agri-Food Canada

Theresa C White

Jeffrey S Tolan

John J Tomashek

Patrick St-Pierre

Nabil Masri

Brian Foody

Christopher D Hindle

Brian R Scott

James A Lavigne

Jennifer Donaldson

Colin Nicholson

Marie-Christine Fournier

Genevieve R Giroux

Katie E A Wallace

Loreta Gudynaite-Savitch

Jas Singh

Daphne Wahnon

Sylvia McHugh

Chao Jiang

Provided are modified cellulase enzymes exhibiting increase cellulose-hydrolyzing activity in the presence of lignin and/or reduced binding to lignin comprising modified linker peptides comprising one or more amino acid substitutions, insertions, or deletions that result in (a) a decrease in the calculated isoelectric point of the linker peptide and/or (b) an increase in the ratio of threonine:serine in the linker peptide relative to a parental linker peptide from which said modified linker peptide is derived. Also provided are genetic constructs comprising nucleic acid sequences encoding for modified cellulase enzymes, methods for the production of the modified cellulase enzymes from host strains and a process for hydrolysing cellulose with the modified cellulases in the presence of lignin.

Cellulase enzymes having a modified linker and reduced lignin binding

Provided are modified cellulase enzymes exhibiting increase cellulose-hydrolyzing activity in the presence of lignin and/or reduced binding to lignin comprising modified linker peptides comprising one or more amino acid substitutions, insertions, or deletions that result in (a) a decrease in the calculated isoelectric point of the linker peptide and/or (b) an increase in the ratio of threonine:serine in the linker peptide relative to a parental linker peptide from which said modified linker peptide is derived. Also provided are genetic constructs comprising nucleic acid sequences encoding for modified cellulase enzymes, methods for the production of the modified cellulase enzymes from host strains and a process for hydrolyzing cellulose with the modified cellulases in the presence of lignin.

A fermentation process for the production of cellulase mixtures is provided. The process comprises providing a genetically modified host filamentous fungus that overexpresses a Xyr1 transcription factor and/or that is partially or completely deficient in expressing one or more hemicellulase enzyme. The host filamentous fungus is cultured in a medium comprising a carbon source. The carbon source contains from about 60 wt % to about 100 wt % hemicellulose-derived carbohydrate and less than 5% of a cellulase-inducing carbohydrate or contains from about 25 wt % to about 100% wt % hemicellulose-derived sugar alcohol in combination with from about 0 wt % to about 75 wt % glucose, glycerol or a combination thereof.

Hosts and fermentation processes for cellulase production

A process for the enzymatic hydrolysis of cellulose to produce a hydrolysis product comprising glucose from a pretreated lignocellulosic feedstock and enzymes for use in the process are provided. The process comprises hydrolyzing an aqueous slurry of a pretreated lignocellulosic feedstock with cellulase enzymes, one or more than one &#x3b2;-glucosidase enzyme and a binding agent for binding the &#x3b2;-glucosidase enzyme to fiber solids present in the aqueous slurry. During the hydrolysis, both the cellulase enzyme and &#x3b2;-glucosidase enzyme bind to the fiber solids. The hydrolysis is performed in a solids-retaining hydrolysis reactor so that unhydrolyzed fiber solids and bound enzyme are retained in the reactor longer than the aqueous phase of the slurry.

Enzyme compositions for the improved enzymatic hydrolysis of cellulose and methods of using same

A process for the enzymatic hydrolysis of cellulose to produce a hydrolysis product comprising glucose from a pretreated lignocellulosic feedstock and enzymes for use in the process are provided. The process comprises partially hydrolyzing an aqueous slurry of a pretreated lignocellulosic feedstock with cellulase enzymes, one or more than one &#x3b2;-glucosidase enzyme and a binding agent for binding the &#x3b2;-glucosidase enzyme to fiber solids present in the aqueous slurry. The unhydrolyzed fiber solids are then separated from the hydrolyzed slurry. The separated fiber solids thus obtained are then re-suspended in an aqueous solution to produce a re-suspended slurry. The hydrolysis is then continued to produce the hydrolysis product comprising glucose.

Enzyme compositions and methods for the improved enzymatic hydrolysis of cellulose

A modified Family 6 cellulase enzyme comprising a proline residue at position 413 is provided. Genetic constructs and genetically modified microbes comprising DNA sequences encoding the modified Family 6 cellulase are also provided. Family 6 cellulases of the invention display improved thermostability, thermophilicity, alkalophilicity, or a combination thereof, relative to the parent Family 6 cellulases. Such cellulases find use in a variety of applications in industry that require cellulase stability and activities at temperatures, pH values, or both, above that of the native enzyme.

Modified cellulases with increased thermostability, thermophilicity, and alkalophilicity

The present invention pertains to a method of converting cellulose to glucose by treating a pretreated lignocellulosic substrate with an enzyme mixture comprising cellulase enzyme and endoglucanase core proteins, wherein the endoglucanase core proteins are present in the enzyme mixture at an amount relative to all endoglucanases from about 35 wt. % to about 100 wt. % and wherein the endoglucanase cellulase enzymes are present in the enzyme mixture at an amount relative to the amount of CBH and EG enzymes from about 2 wt. % to about 50 wt. %. The pretreated lignocellulosic substrate is selected from the group consisting of agricultural residues, residues after starch or sugar removal, dedicated ethanol crops, forestry products, and pulp and paper products, or combinations thereof.

Method for glucose production using endoglucanase core protein for improved recovery and reuse of enzyme

A modified Family 11 xylanase enzyme comprising a sequence that introduces a functional consensus glycosylation site is provided. Non-limiting examples of introduced glycosylation sites include mutation of the amino acid at position 34, 131, 180, 182, or a combination thereof, to an asparagine. The indicated amino acid position in the Family 11 xylanase is determined from sequence alignment of the xylanase of interest with that of axylanase II amino acid sequence. The introduced consensus glycosylation site facilitates increased expression efficiency of the modified xylanase when compared to the expression efficiency of a corresponding xylanase from which the modified xylanase was derived, using similar host strains and growth conditions.

Modified xylanase exhibiting improved expression

Modified xylanases exhibiting improved expression

The present invention pertains to a method of converting cellulose to glucose by treating a pretreated lignocellulosic substrate with an enzyme mixture comprising cellulase enzyme and a modified CBHO, wherein the modified CBHI is present in the enzyme mixture at an amount relative to all CBHI-type enzymes from about 15% to about 100% (w/w),depending upon the modified CBHI used. The pretreated lignocellulosic substrate is selected from the group consisting of agricultural residues, residues after starch or sugar removal dedicated ethanol crops forestry products, and pulp and paper products, or combinations thereof.

Method for glucose production with a modified cellulase mixture

The present invention relates to increasing the production of a protein of interest from a fugal host. The invention discloses nucleotide sequences comprising, a regulatory region in operative association with xylanase secretion sequence and a gene of interest. The gene of interest encodes a protein selected from a pharmaceutical, nutraceutical, industrial, animal feed, food additive and an enzyme. Preferably, the gene of interest encodes a cellulase, hernicellulase, a lignin degrading enzyme, pectinase, protease, or peroxidase. The present invention also relates to vectors and hosts comprising these nucleic acid sequences, and to methods for the production of a protein of interest.

Enhanced expression of proteins in genetically modified fungi

This invention relates to the genetic modification of a microbe to enhance its production of an enzyme, beta-glucosidase, that is important in the cellulose conversion process. The inventors have discovered genetic constructs that, when expressed in recombinant microbes, dramatically increase the amount of beta-glucosidase produced relative to untransformed microbes. The genetic constructs comprise a promoter, a xylanase secretion signal, and a mature beta-glucosidase coding region. The increased level of beta-glucosidase significantly increases the efficiency of hydrolysis of cellulose to glucose by cellulase enzymes, thereby enhancing the production of fuel ethanol from cellulose.

Genetic constructs and genetically modified microbes for enhanced

During the enzymatic 'stone washing' of a denim fabric and/or garments, an undesirable redeposition of blue dye often occurs on the surfaces of the denim. The invention relates to a means of overcoming this problem using an enzyme composition comprised of Trichoderma endoglucanases and Trichoderma cellobiohydrolases that has been partially digested by a protease enzyme to separate its core and binding domains. The use of this composition reduces the redeposition of the blue dye and hence improves the stone washing process relative to using a redepositing or backstaining cellulase.

Protease-Treated and purified cellulase compositions and methods for

A 1.2 kb fragment of the 5' regulatory region (from bp -1107 to +100) was fused to the GUS (.beta.-glucuronidase) reporter gene and BN115-promoted GUS expression was observed in green tissues of transgenic Brassica napus plants only after incubation at 2.degree. C. No expression was observed after incubation at 22.degree. C., either in the presence or absence of abscisic acid. Microprojectile bombardment of winter B. napus leaves with a BN115 promoter/GUS construct yielded similar results and was used to analyze a series of deletions from the 5' end of the promoter. Results obtained from transient expression studies showed that the low temperature regulation of BN115 expression involves a possible enhancer region between bp -1107 and -606 and a second positive regulatory region located between bp -302 and -274. Deletion analyses and results from replacement with a truncated CaMV (cauliflower mosaic virus) 35S promoter suggest that the minimal size required for any maintenance of low temperature GUS expression is a -300 bp fragment. Within this fragment are two 8 bp elements with the sequence TGGCCGAC, which are identical to those present in the positive regulatory region of the promoter of the homologous Arabidopsis cor15a gene and to a 5 bp core sequence in the low temperature- and dehydration-responsive elements (LTREs and DREs) identified in the promoter regions of several cold-responsive Arabidopsis thaliana genes. Mutation of either one or both of the GGCAC core sequence of the putative LTRE's to AATTC resulted in loss of cold-inducible gene expression, providing for the first time, direct evidence that CCGAC is required for low temperature expression. Furthermore, replacement of an enhancer region (-605 to -1107) of the promoter with a more active enhancer from the 35S constitutive cauliflower mosaic virus (CaMV) promoter resulted in a 'hybrid' promoter with increased low temperature induced activity several fold over that of the native promoter.

Growing community of inventors

Ottawa, Canada

Theresa C White