(264a) Site-Directed Mutagenesis of Family 64 CBM Provides Insights into the Anomalous Binding Interactions with Pretreated Cellulose during Biomass Saccharification

Lignocellulosic biomass, composed of cellulose, hemicellulose and lignin, is broken down into simple fermentable sugars in nature by cellulolytic enzyme cocktails secreted by various microbes. Enzymatically-catalyzed breakdown of cellulose into sugars like glucose has critical significance towards cost-effective production of cellulosic biofuels. Lignocellulosic biomass pretreatment methods can increase the enzymatic accessibility of cellulose microfibrils and/or change the crystalline ultrastructure of cellulose that can also impact enzyme activity (1). Cellulose III, an unnatural allomorph of cellulose, is formed during anhydrous liquid ammonia based pretreatment of the native crystalline cellulose I allomorphic state (1-2). Cellulose III has already been shown to have anomalous binding interactions with carbohydrate-binding modules and cellulolytic enzymes (e.g., cellulases) in our previous studies (3). Here, we report results from our site-directed mutagenesis study of a novel carbohydrate-binding module (CBM) belonging to family 64, followed by detailed binding characterization of the mutant CBM library with both cellulose I and cellulose III. These studies shed light into the structure-function relationships guiding the binding of native and engineered CBMs to unnatural cellulose III, as opposed to native cellulose I. This work has direct implications on the cost-effective enzymatic hydrolysis of biomass into fermentable sugars in lignocellulosic biorefineries employing pretreatments that can modify the cellulose ultrastructure.

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2. da Costa Sousa, L. et al. Next-generation ammonia pretreatment enhances cellulosic biofuel production. Energy Environ. Sci. 9, 1215–1223 (2016).

3. Gao, D. et al. Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis. Proc. Natl. Acad. Sci. 110, 10922–10927 (2013).