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Understanding and Engineering Cellulase Binding to Biomass Components

  • Author(s): Strobel, Kathryn Lynn
  • Advisor(s): Clark, Douglas S
  • et al.
Abstract

Lignocellulosic biomass is an abundant, low-cost resource for the renewable production of fuels and chemicals. To unlock the potential of lignocellulosic biomass, the cellulose must be broken down into sugars before fermentation to produce ethanol, butanol, or other bio-based products. Unfortunately, lignocellulose is highly resistant to enzymatic degradation, necessitating high enzyme loadings that increase the cost of biofuels. The recalcitrance of biomass stems in part from the presence of lignin, a major component of lignocellulosic biomass. Lignin impedes enzymatic hydrolysis by non-productively binding cellulases and contributing to cellulase denaturation. Despite numerous studies documenting cellulase adsorption to lignin, the structural basis has not been fully elucidated and few attempts have been made to engineer enzymes for reduced lignin affinity.

In this work, we investigate and engineer cellulase adsorption to lignin and the resulting effect on hydrolysis of cellulose. The lignin inhibition of two homologous cellulases, T. reesei Cel7A and T. emersonii Cel7A, was found to differ significantly. In Chapter 2, we propose that differences in surface charge, stability, and glycosylation patterns may be the driving force/s behind the observed differences in lignin inhibition and we suggest engineering strategies for improving lignin tolerance of Cel7A catalytic domains.

Chapters 3 and 4 detail our efforts to investigate the mechanisms of cellulase lignin adsorption and engineer an enzyme with reduced lignin affinity using site directed mutagenesis of the T. reesei Cel7A carbohydrate binding module (CBM) and linker. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for the majority of lignin affinity. Cellulose and lignin affinity of the alanine mutants were highly correlated, indicating similar binding mechanisms for cellulose and lignin. CBM mutations that added hydrophobic or positively charged residues decreased the selectivity toward cellulose, while mutations that added negatively charged residues increased the selectivity. Mutating the linker to alter predicted glycosylation patterns greatly impacted lignin affinity but did not affect cellulose affinity. Beneficial mutations were combined to generate a mutant with 2.5 fold less lignin affinity and fully retained cellulose affinity. This mutant was not inhibited by added lignin during hydrolysis of Avicel and generated 40% more glucose than the wild type enzyme from dilute acid-pretreated Miscanthus. The mutations studied here inform engineering efforts of other homologous CBMs and will hopefully contribute to reducing the cost of biofuels.

The final chapter details the development of a high-throughput selection platform for engineering protease enzymes with new sequence specificity. Proteases are commonly used in research, industry, and medicine, and there is considerable promise for new proteases that could cleave at a user-specified sequence. Positive selection and counter-selection were combined to select a tobacco etch virus protease mutant with new substrate compatibility.

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