Alliance for Sustainable Energy, LLC

Midwest Research Institute

Alliance for Sustainably Enegry, LLC

Stephen Robert Decker

Michael E Himmel

William S Adney

John O Baker

Todd B Vinzant

Shi-You Ding

Gregg Tyler Beckham

Jeffrey G Linger

Eric E Jarvis

Yat-Chen Chou

Arjun Singh

Qi Xu

Kara Podkaminer

Larry Taylor, Ii

Venkataramanan Subramanian

Mark R Nimlos

Eric P Knoshaug

Roman Brunecky

Suzanne Lantz McCarter

Suzanne Mc Carter

Raphael Nieves

Michael F Crowley

Tina Jeoh

Disclosed herein are methods of making multifunctional microbial cellulases. The engineered multifunctional microbial cellulases disclosed herein exhibit improved activity over native cellulases.

Multifunctional microbial cellulases

Disclosed herein are chimeric Cel7A polypeptides useful for producing biofuels from lignocellulosic biomass.

Fungal cellobiohydrolases

Nucleic acid sequences encoding chimeric polypeptides that exhibit enhanced cellulase activities are disclosed herein. These nucleic acids may be expressed in hosts such as fungi, which in turn may be cultured to produce chimeric polypeptides. Also disclosed are chimeric polypeptides and their use in the degradation of cellulosic materials.

Enhanced processive cellulases

Provided herein is an isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide, wherein the mutations reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. Also provided herein is an isolated Cel7A polypeptide comprising increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The increased O-linked glycosylation is a result of the addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide. In some embodiments, the isolated Cel7A polypeptide comprising mutations in the catalytic domain of the polypeptide relative to the catalytic domain of a wild type Cel7A polypeptide further comprises increased O-linked glycosylation of the linker domain relative to a linker domain of a wild type Cel7A polypeptide. The mutations in the catalytic domain reduce N-linked glycosylation of the isolated polypeptide relative to the wild type polypeptide. The addition of and/or substitution of one or more serine and/or threonine residues to the linker domain relative to the linker domain of the wild type polypeptide increases O-linked glycosylation of the isolated polypeptide. Further provided are compositions comprising such polypeptides and nucleic acids encoding such polypeptides. Still further provided are methods for making such polypeptides.

Cellobiohydrolase I enzymes

Purified cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7A)) enzymes fromdemonstrate a high level of specific performance in comparison to other Cel7 family member enzymes when formulated with purified EIcd endoglucanase fromand tested on pretreated corn stover. This result is true of the purified native enzyme, as well as recombinantly expressed enzyme, for example, that enzyme expressed in a non-nativehost. In a specific example, the specific performance of the formulation using purified recombinant Cel7A fromexpressed inis increased by more than 200% when compared to a formulation using purified Cel7A from

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Purified cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7A) enzymes fromdemonstrate a high level of specific performance in comparison to other Cel7 family member enzymes when formulated with purified EIcd endoglucanase fromand tested on pretreated corn stover. This result is true of the purified native enzyme, as well as recombinantly expressed enzyme, for example, that enzyme expressed in a non-nativehost. In a specific example, the specific performance of the formulation using purified recombinant Cel7A fromexpressed inis increased by more than 200% when compared to a formulation using purified Cel7A from

The invention provides a thermal tolerant cellulase that is a member of the glycoside hydrolase family. The invention further discloses this cellulase as Gux1. Gux1 has been isolated and characterized from. The invention further provides recombinant forms of the identified Gux1. Methods of making and using Gux1 polypeptides, including fusions, variants, and derivatives, are also disclosed.

The disclosure provides a method for preparing an active exoglucanase in a heterologous host of eukaryotic origin. The method includes mutagenesis to reduce glycosylation of the exoglucanase when expressed in a heterologous host. It is further disclosed a method to produce variant cellobiohydrolase that is stable at high temperature through mutagenesis.

Cellobiohydrolase I gene and improved variants

The invention provides a thermal tolerant cellulase that is a member of the glycoside hydrolase family. The invention further discloses this cellulase as GuxA. GuxA has been isolated and characterized fromThe invention further provides recombinant forms of the identified GuxA. Methods of making and using GuxA polypeptides, including fusions, variants, and derivatives, are also disclosed.

A process for production of an alkaline tolerant dextranase enzyme comprises culturing a dextran-producing microorganism having accession no. ATCC PTA-3866 to produce an alkaline tolerant dextranase, Dex 1 wherein the protein in said enzyme is characterized by a MW of 63.3 kDa and Dex 2 wherein its protein is characterized by a MW of 81.8 kDa.

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Berthoud, CO, United States of America

Stephen Robert Decker