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Open Access Publications from the University of California

Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2014)

The transition from the production of fuels and chemicals from fossil sources to cellulosic feedstocks is an important step towards a more carbon-neutral economy. Cellulose is the most abundant polymer on the planet. Its constituent beta-1,4 linked glucose units, when deconstructed to glucose monomers, can be fermented into ethanol, butanol, and other next-generation renewable products. Biomass costs less on an energy basis than oil, and some cellulosic crops require far fewer water and nitrogen inputs than corn-based bioethanol production. However, technical challenges remain, and cellulosic sugar is not yet a major platform for fuel and chemical production. The high cost of cellulolytic enzyme production, the large quantities of enzyme needed to complete the transformation of cellulose to glucose, and the presence of lignin in the heterogeneous biomass are a few major barriers to the large-scale production of cellulose-derived sugar.

Lignin inhibition via nonproductive binding of cellulase enzymes to the lignin surface decreases the overall efficiency of cellulose hydrolysis, and precludes recycling of lignin-bound enzymes. Although nonproductive binding of cellulases to lignin has been observed for many years, efforts to characterize the kinetics and structural drivers of this interaction have been lacking. In this work, lignin was isolated and characterized from acid-pretreated Miscanthus, in an attempt to isolate a lignin that is chemically similar to that present in an industrially relevant feedstock. Lignin films were spin-cast and characterized, in order to produce a flat, homogeneous substrate for surface binding assays. Cellulase enzymes were purified from native or heterologous expression hosts, and quartz crystal microgravimetry with dissipation monitoring (QCM-D) was used to measure the adsorption, desorption, and irreversible adsorption rates to the lignin surface. Using these tools, the several different reaction mechanisms of lignin-cellulase binding were evaluated. Although most existing lignin-binding literature uses Langmuir isotherms to quantify lignin-cellulase interactions, the data show that the mechanism that best fits the measured data is a transition model with multiple binding sites. Methods for comparing kinetic mechanisms and calculating kinetic parameters are presented.

The cellulose-degrading system of Hypocrea jecorina is well known as an industrial stan- dard for enzymatic biomass hydrolysis. In this work, the lignin-binding kinetics of the four most important H. jecorina cellulases have been characterized. Existing literature shows that the carbohydrate binding domain is important in lignin binding. In order to quantify this effect, full-length enzymes and their isolated catalytic domains have been tested for their lignin-binding kinetics. While CBMs are responsible for fast binding of cellulases to lignin, our work implicates the catalytic domain in the irreversible aspect of lignin-binding kinet- ics. Furthermore, several-fold differences in adsorption rates between homologous CBMs are noted, as well as nearly 10-fold differences in adsorption rates between two homologous catalytic domains. Using this technique, we have uncovered new targets for evolution of cel- lulases. Finally, future directions in the study of lignin-cellulase interactions are discussed.

The final chapter in this work is an attempt to integrate lignin oxidation via oxidative enzymes into an ionic liquid pretreatment process. Certain ionic liquids are efficient solvents for whole biomass, and furthermore are able to cleave non phenolic lignin model dimer compounds, provided the dimers have been oxidized at the alpha-hydroxyl position. An effort to extend these model compound results to lignin oxidation and breakdown is reported.

Lignin is both an energy-dense biomass component and potential source of aromatic com- pounds, and a physical and competitive inhibitor of cellulase enzymes. This thesis presents efforts to decrease the cost of sugar production, both through the study of nonproductive cellulase-interactions, and through attempts to modify lignin structure for the extraction of value-added products.

    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2015)
    The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. 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 lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs.
      Cover page: Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin*
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2020)
      Single-cell CRISPR screens enable the exploration of mammalian gene function and genetic regulatory networks. However, use of this technology has been limited by reliance on indirect indexing of single-guide RNAs (sgRNAs). Here we present direct-capture Perturb-seq, a versatile screening approach in which expressed sgRNAs are sequenced alongside single-cell transcriptomes. Direct-capture Perturb-seq enables detection of multiple distinct sgRNA sequences from individual cells and thus allows pooled single-cell CRISPR screens to be easily paired with combinatorial perturbation libraries that contain dual-guide expression vectors. We demonstrate the utility of this approach for high-throughput investigations of genetic interactions and, leveraging this ability, dissect epistatic interactions between cholesterol biogenesis and DNA repair. Using direct capture Perturb-seq, we also show that targeting individual genes with multiple sgRNAs per cell improves efficacy of CRISPR interference and activation, facilitating the use of compact, highly active CRISPR libraries for single-cell screens. Last, we show that hybridization-based target enrichment permits sensitive, specific sequencing of informative transcripts from single-cell RNA-seq experiments.
        Cover page: Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing
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