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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Lignin impairs Cel7A degradation of in vitro lignified cellulose by impeding enzyme movement and not by acting as a sink

Abstract Background Cellulose degradation by cellulases has been studied for decades due to the potential of using lignocellulosic biomass as a sustainable source of bioethanol. In plant cell walls, cellulose is bonded together and strengthened by the polyphenolic polymer, lignin. Because lignin is tightly linked to cellulose and is not digestible by cellulases, is thought to play a dominant role in limiting the efficient enzymatic degradation of plant biomass. Removal of lignin via pretreatments currently limits the cost-efficient production of ethanol from cellulose, motivating the need for a better understanding of how lignin inhibits cellulase-catalyzed degradation of lignocellulose. Work to date using bulk assays has suggested three possible inhibition mechanisms: lignin blocks access of the enzyme to cellulose, lignin impedes progress of the enzyme along cellulose, or lignin binds cellulases directly and acts as a sink. Results We used single-molecule fluorescence microscopy to investigate the nanoscale dynamics of Cel7A from Trichoderma reesei , as it binds to and moves along purified bacterial cellulose in vitro. Lignified cellulose was generated by polymerizing coniferyl alcohol onto purified bacterial cellulose, and the degree of lignin incorporation into the cellulose meshwork was analyzed by optical and electron microscopy. We found that Cel7A preferentially bound to regions of cellulose where lignin was absent, and that in regions of high lignin density, Cel7A binding was inhibited. With increasing degrees of lignification, there was a decrease in the fraction of Cel7A that moved along cellulose rather than statically binding. Furthermore, with increasing lignification, the velocity of processive Cel7A movement decreased, as did the distance that individual Cel7A molecules moved during processive runs. Conclusions In an in vitro system that mimics lignified cellulose in plant cell walls, lignin did not act as a sink to sequester Cel7A and prevent it from interacting with cellulose. Instead, lignin both blocked access of Cel7A to cellulose and impeded the processive movement of Cel7A along cellulose. This work implies that strategies for improving biofuel production efficiency should target weakening interactions between lignin and cellulose surface, and further suggest that nonspecific adsorption of Cel7A to lignin is likely not a dominant mechanism of inhibition.

09 BIOMASS FUELS↗
DOE OSTI2024

Single-molecule tracking reveals dual front door/back door inhibition of Cel7A cellulase by its product cellobiose

Degrading cellulose is a key step in the processing of lignocellulosic biomass into bioethanol. Cellobiose, the disaccharide product of cellulose degradation, has been shown to inhibit cellulase activity, but the mechanisms underlying product inhibition are not clear. We combined single-molecule imaging and biochemical investigations with the goal of revealing the mechanism by which cellobiose inhibits the activity of Trichoderma reesei Cel7A, a well-characterized exo-cellulase. We find that cellobiose slows the processive velocity of Cel7A and shortens the distance moved per encounter; effects that can be explained by cellobiose binding to the product release site of the enzyme. Cellobiose also strongly inhibits the binding of Cel7A to immobilized cellulose, with a K i of 2.1 mM. The isolated catalytic domain (CD) of Cel7A was also inhibited to a similar degree by cellobiose, and binding of an isolated carbohydrate-binding module to cellulose was not inhibited by cellobiose, suggesting that cellobiose acts on the CD alone. Finally, cellopentaose inhibited Cel7A binding at micromolar concentrations without affecting the enzyme’s velocity of movement along cellulose. Together, these results suggest that cellobiose inhibits Cel7A activity both by binding to the “back door” product release site to slow activity and to the “front door” substrate-binding tunnel to inhibit interaction with cellulose. These findings point to strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

BETO 2021 Peer Review - Enzyme Engineering and Optimization (EEO)

The FY2020 SOT economic model for the DMR BDO process indicates that the production and use of biomass degrading enzymes represents ~10% of the MFSP of BDO. We are thus working to enhance the performance of the dominant cellulase enzyme, Cel7A. In Fy2018-19, we selected 100 promising genes from the "Cel7A wheel of life" and cloned them into T. reesei using a constitutive promotor. This natural diversity screening resulted in the discovery of several enzymes exhibiting improved characteristics relative to the industry standard Cel7A from T. reesei (Tr). Growth of the transformed host on glucose insured that the Cre1 induced cellulase expression cascade was suppressed, thus enabling purification of the target gene product. The first top performing enzyme found was from P. funiculosum (Pf). Other top performing enzymes were identified from T. aculeatus, T. terrestris, and A. oryzae. Computational modeling predicted the structural subsites responsible for the improvements, which were cloned into the sequence of PfCel7A. The recombinant enzymes were expressed in T. reesei, purified, and tested. Testing was not possible with SOT relevant substrates, so the best-case substrate was used; solids from the DMR process subjected to a novel dilute alkaline wash to remove the "lignin shield." Two chimeric Cel7A enzymes show improved performance (1.2 to 1.35x) relative to PfCel7A, which in turn showed considerable improvement (1.6x) relative to T. reesei Cel7A. These new enzymes have been provided to our industrial partner for evaluation.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗
DOE OSTI2025

Advanced Cellobiohydrolases (CRADA Final Report)

The purpose of the TCF project was to develop and transfer advanced Cel6A and Cel7A cellulase enzymes to Novozymes for testing on NREL provided DMR treated corn stover. In both cases, we developed advanced Cel6A and Cel7A enzymes that outperform the baseline T. reesei enzymes that are commonly available. The final milestone was to demonstrate an 80% glucan conversion on DMR treated corn stover with an enzyme loading of 10 mg/g glucan. This goal was achieved relatively early in the project and a new stretch goal for Novozymes became to test these enzymes in the Gen 1.5 yeast fermentation platform. The outcome was marginally successful due to temperature and pH optima mismatches between the enzymes (initially developed for use in a standard enzyme cocktail) and conditions suitable for yeast fermentation. Covid-19 and several re-organizations at Novozymes did significantly impact the project timeline, extending it to 2023. However, all of the original goals of the were successfully met or exceeded.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

Dissociation Mechanism of Processive Cellulases Explored Through Molecular Simulation

Cellulose is the most abundant biopolymer on the planet, with significant potential as a feedstock for bioderived products. In nature, fungi and bacteria secrete cellulases to hydrolyze cellulose into smaller carbohydrates for incorporation into their own metabolic processes. For effective biomass utilization at industrial scale, these cellulases are coupled together to produce a clean sugar stream. One such cellulase is Cel7A from Trichoderma reesei, which is used extensively in biotechnological applications. However, dissociation of Cel7A from a bound cellulose strand is rate-limiting, reducing catalytic efficiency and yields within industrial applications. To explore this unknown dissociation mechanism and devise potential enzyme engineering strategies to mitigate slow dissociation, we conduct Hamiltonian replica exchange molecular dynamics simulations. Two postulated dissociation mechanisms were tested ('dethreading' of the bound cellulose strand from the binding tunnel and 'clamshell'-like opening of the binding tunnel and recrystallization of the bound cellulose). For each mechanism, free energy profiles were determined together with estimated diffusion coefficients. The kinetics for both mechanisms were then estimated and compared against experimentally measured dissociation rates, which suggest that the dethreading mechanism ought to be preferred. These findings are further supported by dissociation rates measured on engineered mutants incapable of undergoing the clamshell mechanism.

09 BIOMASS FUELS↗
DOE OSTI2019

The Dissociation Mechanism of Processive Cellulases

Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing. We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.

09 BIOMASS FUELS↗
DOE OSTI2021

Integrated multi-wavelength microscope combining TIRFM and IRM modalities for imaging cellulases and other processive enzymes

We describe a multimodal microscope for visualizing processive enzymes moving on immobilized substrates. The instrument combines interference reflection microscopy (IRM) with multi-wavelength total internal reflectance fluorescence microscopy (TIRFM). The microscope can localize quantum dots with a precision of 2.8 nm at 100 frames/s, and was used to image the dynamics of the cellulase, Cel7a interacting with surface-immobilized cellulose. The instrument, which was built with off-the-shelf components and is controlled by custom software, is suitable for tracking other degradative enzymes such as collagenases, as well as motor proteins moving along immobilized tracks.

09 BIOMASS FUELS↗
DOE OSTI2021

Chimeric cellobiohydrolase I expression, activity, and biochemical properties in three oleaginous yeast

Consolidated bioprocessing using oleaginous yeast is a promising modality for the economic conversion of plant biomass to fuels and chemicals. However, yeast are not known to produce effective biomass degrading enzymes naturally and this trait is essential for efficient consolidated bioprocessing. We expressed a chimeric cellobiohydrolase I gene in three different oleaginous, industrially relevant yeast: Yarrowia lipolytica, Lipomyces starkeyi, and Saccharomyces cerevisiae to study the biochemical and catalytic properties and biomass deconstruction potential of these recombinant enzymes. Our results showed differences in glycosylation, surface charge, thermal and proteolytic stability, and efficacy of biomass digestion. L. starkeyi was shown to be an inferior active cellulase producer compared to both the Y. lipolytica and S. cerevisiae enzymes, whereas the cellulase expressed in S. cerevisiae displayed the lowest activity against dilute-acid-pretreated corn stover. Comparatively, the chimeric cellobiohydrolase I enzyme expressed in Y. lipolytica was found to have a lower extent of glycosylation, better protease stability, and higher activity against dilute-acid-pretreated corn stover.

59 BASIC BIOLOGICAL SCIENCES↗