Iogen Energy Corporation

James A Lavigne

John J Tomashek

Brian R Scott

Patrick St-Pierre

Nabil Masri

Theresa C White

Christopher Hill

Annie Tremblay

Martine Whissel

Loreta Gudynaite-Savitch

Christopher D Hindle

Chengsong Liu

Christopher M D Hill

Fuad Tahna

Jacqueline Montalibet

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 isolated cellobiohydrolases comprising a modified Glycoside Hydrolase (GH) Family 7 catalytic domain, a GH Family 7 catalytic domain and a modified Family 1 carbohydrate binding module (CBM), or both a modified Family 7 catalytic domain and a modified Family 1 CBM. Such isolated cellobiohydrolases exhibit from 45% to about 99.9% amino acid sequence identity to amino acids 1-436 of SEQ ID NO: 1 or to amino acids 1-438 of SEQ ID NO: 2 and improved activity on process substrates. Also provided are genetic constructs and genetically modified microbes for expressing the isolated cellobiohydrolases, a process for producing the isolated cellobiohydrolases, cellulase enzyme mixtures comprising the isolated cellobiohydrolase and a process for hydrolyzing a cellulosic substrate with such cellulase enzyme mixtures.

Cellobiohydrolase enzymes

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.

Provided are modified beta-glucosidase enzymes, derived from theCel3A beta-glucosidase, that exhibit improvements in one or more kinetic parameters (K, K, k) comprising amino acid substitutions at one or more of positions 43, 101, 260 and 543. Also provided are genetic constructs comprising nucleotide sequences encoding for modified beta-glucosidase enzymes, methods for the production of modified beta-glucosidase enzymes from host strains and the use of the modified beta-glucosidase enzymes in the hydrolysis of cellulose.

Beta-glucosidase enzymes

A modified Family 6 glycosidase enzyme comprising amino acid substitutions at one or more positions selected from the group 182, 367, 399, 400 and 427 is provided (the position determined form alignment of a parental Family 6 glycosidase with SEQ ID NO: 1). Genetic constructs and genetically modified microbes comprising nucleic sequences encoding the modified Family 6 glycosidase are also provided. Family 6 glycosidase of the invention display decreased hydrolysis activity of beta 1-4 linked polysaccharides and increased hydrolysis activity of beta 1-3, 1-4 linked polysaccharides compared with a parental Family 6 glycosidase. Such glycosidases find use in a variety of applications in industry, e.g., in hydrolysis of beta 1-3, 1-4 linked polysaccharides during the processing of cereal grains or the production of alcohol, animal feed or food products.

Modified family 6 glycosidases with altered substrate specificity

Provided are modified beta-glucosidase enzymes, derived from theCel3A beta-glucosidase, that exhibit improved stability at low pH, low pH and high aeration, low pH and high agitation, or low pH and elevated temperature. Also provided are genetic constructs comprising nucleotide sequences encoding for modified beta-glucosidase enzymes, methods for the production of modified beta-glucosidase enzymes from host strains and the use of the modified beta-glucosidase enzymes in the hydrolysis of cellulose.

Modified beta-glucosidases with improved stability

A modifiedFamily 6 (TrCel6A) cellulase enzyme comprising amino acid substitutions at one or more positions selected from the group consisting of 129, 322, 363 and 410 of SEQ ID NO: 1 is provided. Genetic constructs and genetically modified microbes comprising nucleic sequences encoding the modified TrCel6a cellulase are also provided. The modified TrCel6A cellulase of the invention display at least a 15% decrease in inactivation by lignin relative to a parental TrCel6A cellulase from which the modified TrCel6A is derived. Such cellulases find use in a variety of applications in industry requiring enzymatic hydrolysis of cellulose in the presence of lignin, e.g., the hydrolysis of pretreated lignocellulosic feedstocks for the production of fermentable sugars, sugar alcohols and fuel alcohols.

Family 6 cellulase with decreased inactivation by lignin

A Family 6 cellulase variant enzyme comprising one or more than one amino acid substitution selected from a basic, polar or non-polar amino acid at position 103, a valine or isoleucine at position 136, a tyrosine at position 186, a glutamic acid or glutamine at position 365 and a glutamine at position 410 is provided (said position determined form alignment of the parental Family 6 with SEQ ID NO: 1). Genetic constructs and genetically modified microbes comprising DNA sequences encoding the Familycellulase variant are also provided. Family 6 cellulases of the invention display reduced inhibition by glucose relative to the parent Family 6 cellulases. Such cellulases find use in a variety of applications in industry, e.g., in the hydrolysis of pretreated lignocellulosic feedstock, that require cellulose activity in the presence glucose concentrations that would otherwise inhibit the activity of the parental enzyme.

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Nepean, Canada

James A Lavigne