Publications

Authors: Agrawal, A., Bandi, C. K., Burgin, T., Woo, Y., Mayes, H. B., and Chundawat, S. P. S.

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Abstract: Engineering of carbohydrate-active enzymes like glycosylases for chemoenzymatic synthesis of bespoke oligosaccharides has been limited by the lack of suitable directed evolution-based protein engineering methods. Currently, there are no ultrahigh-throughput screening methods available for rapid and highly sensitive single-cell-based screening of evolved glycosynthase enzymes employing azido sugars as substrates. Here, we report a fluorescence-based approach employing click-chemistry for the selective detection of glycosyl azides (versus free inorganic azides) that facilitated ultrahigh-throughput in-vivo single cell-based assay of glycosynthase activity. This discovery has led to the development of a directed evolution methodology for screening and sorting glycosynthase mutants for the synthesis of desired fucosylated oligosaccharides. Our screening technique facilitated rapid fluorescence-activated cell sorting of a large library of glycosynthase variants (>106 mutants) expressed in E. coli to identify several novel mutants with increased activity for β-fucosyl-azide activated donor sugars towards desired acceptor sugars, demonstrating the broader applicability of this methodology.

Authors: Nemmaru, B., Ramirez, N., Farino, C. J., Yarbrough, J. M., Kravchenko, N., and Chundawat, S. P. S.

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Abstract: Dissociation of non-productively bound cellulolytic enzymes from cellulose is hypothesized to be a key rate-limiting factor impeding cost-effective biomass conversion to fermentable sugars. However, the role of carbohydrate-binding modules (CBMs) in enabling nonproductive enzyme binding is not well understood. Here, we examine the subtle interplay of CBM binding and cellulose hydrolysis activity for three models of type-A CBMs (Families 1, 3a, and 64) tethered to multifunctional endoglucanase (CelE) on two distinct cellulose allomorphs (i.e., cellulose I and III). We generated a small library of mutant CBMs with varying cellulose affinity, as determined by equilibrium binding assays, followed by monitoring the cellulose hydrolysis activity of CelE-CBM fusion constructs. Finally, kinetic binding assays using quartz crystal microbalance with dissipation were employed to measure CBM adsorption and desorption rate constants k_on and k_off, respectively, towards nanocrystalline cellulose derived from both allomorphs. Overall, our results indicate that reduced CBM equilibrium binding affinity towards cellulose I alone, resulting from increased desorption rates k_off and reduced effective adsorption rates (n * k_on), is correlated to overall improved endocellulase activity. Future studies could employ similar approaches to unravel the role of CBMs in nonproductive enzyme binding and develop improved cellulolytic enzymes for industrial applications.

Authors: Chundawat, S. P. S.*, Pal, R., Zhao, C., Campbell, T., Teymouri, F., Videto, J., Nielson, C., Wieferich, B., Sousa, L., Dale, B. E., Balan, V., Chipkar, S., Aguado, J., Burke, E., and Ong, R. G.

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Abstract: Lignocellulosic materials are plant-derived feedstocks, such as crop residues (e.g., corn stover, rice straw, and sugar cane bagasse) and purpose-grown energy crops (e.g., miscanthus, and switchgrass) that are available in large quantities to produce biofuels, biochemicals, and animal feed. Plant polysaccharides (i.e., cellulose, hemicellulose, and pectin) embedded within cell walls are highly recalcitrant towards conversion into useful products. Ammonia fiber expansion (AFEX) is a thermochemical pretreatment that increases accessibility of polysaccharides to enzymes for hydrolysis into fermentable sugars. These released sugars can be converted into fuels and chemicals in a biorefinery. Here, we describe a laboratory-scale batch AFEX process to produce pretreated biomass on the gram-scale without any ammonia recycling. The laboratory-scale process can be used to identify optimal pretreatment conditions (e.g., ammonia loading, water loading, biomass loading, temperature, pressure, residence time, etc.) and generates sufficient quantities of pretreated samples for detailed physicochemical characterization and enzymatic/microbial analysis. The yield of fermentable sugars from enzymatic hydrolysis of corn stover pretreated using the laboratory-scale AFEX process is comparable to pilot-scale AFEX process under similar pretreatment conditions. This paper is intended to provide a detailed standard operating procedure for the safe and consistent operation of laboratory-scale reactors for performing AFEX pretreatment of lignocellulosic biomass.

Authors: Chundawat, S. P. S.

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Abstract: Biochemical conversion of plant-based insoluble carbohydrate polymers, such as starch from corn grains or cellulose-hemicellulose from corn stover, into soluble fermentable sugars (e.g., glucose and xylose) for bioenergy production has seen tremendous research activity and commercial-scale biorefineries deployment over the last three decades, particularly in regions around the world that have a dominant agricultural-based economy. Therefore, educators in schools and universities have developed various hands-on experimental activities to engage the general public and students either in outreach events or lab/classroom-based settings to instruct students on various inter-disciplinary concepts relevant to bioenergy and biochemicals production. One of the limitations of most available protocols is the lack of systematic and comprehensive comparison of educator-friendly analytical tools and protocols for quantitative analysis of water-soluble carbohydrates commonly encountered in a biorefinery backdrop during the biochemical conversion of lignocellulosic biomass to biofuels/biochemicals. Here, we systematically compare and validate findings from four leading analytical approaches for detection and quantification of lignocellulosic biomass derived soluble carbohydrates. We compare these assay methods based on the overall ease of use, detection accuracy/sensitivity, commercial availability, analytical cost per assay run, and suitability for use by instructors in biorefining specific hands-on activity protocols. Next, we provide a detailed instructional protocol that utilizes one of these validated soluble sugar assays as part of a ∼90 min hands-on bioenergy focused activity (called ‘Grass-to-Gas!’) conducted at Rutgers University with pre-university high school students. ‘Grass-to-Gas!’ activity involves students running biochemical assays that helps them understand the various facets of cellulosic biomass hydrolysis by commercial cellulase enzymes and monitoring the total glucose product released using our validated sugar assays to finally estimate the fractional conversion of cellulose-to-glucose. Lastly, we further demonstrate how such carbohydrate-based analytical methods can be used by instructors to help university students explore and understand various chemistry, biochemistry, and chemical engineering concepts relevant to other advanced operations involved in lignocellulose biorefining. These activity protocols would greatly aid educators teaching interdisciplinary science and engineering concepts to students in the backdrop of lignocellulose biorefining.

Authors: Chundawat, S. P. S.*, Nemmaru, B., Hackl, M., Brady, S. K., Hilton, M. A., Johnson, M. M., Chang, S., Lang, M. J., Huh, H., Lee, S.-H., Yarbrough, J. M., López, C. A., and Gnanakaran, S.

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Abstract: Cellulolytic microorganisms, like Trichoderma reesei or Clostridium thermocellum, frequently have non-catalytic carbohydrate-binding modules (CBMs) associated with secreted or cell surface bound multidomain carbohydrate-active enzymes (CAZymes) like cellulases. Mostly type-A family CBMs are known to promote cellulose deconstruction by increasing the substrate-bound concentration of cognate cellulase catalytic domains. However, due to the interfacial nature of cellulose hydrolysis and the structural heterogeneity of cellulose, it has been challenging to fully understand the role of CBMs on cellulase activity using classical protein-ligand binding assays. Here, we report a single-molecule CAZyme assay for an industrially relevant processive cellulase Cel7A (from T. reesei) to reveal how subtle CBM1 binding differences can drastically impact cellulase motility/velocity and commitment to initial processive motion for deconstruction of two well-studied crystalline cellulose allomorphs (namely cellulose I and III). We take a multifaceted approach to characterize the complex binding interactions of all major type-A family representative CBMs including CBM1, using an optical-tweezers based single-molecule CBM-cellulose bond ‘rupture’ assay to complement several classical bulk ensemble protein-ligand binding characterization methods. While our work provides a basis for the ‘cautious’ use of Langmuir-type adsorption models to characterize classical protein-ligand binding assay data, we highlight the critical limitations of using such overly simplistic models to gain a truly molecular-level understanding of interfacial protein binding interactions at heterogeneous solid-liquid interfaces. Finally, molecular dynamics simulations provided a theoretical basis for the complex binding behavior seen for CBM1 towards two distinct cellulose allomorphs reconciling experimental findings from multiscale analytical methods.

Authors: Bandi, C. K., Goncalves, A., Pingali, S. V., and Chundawat, S. P. S.

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Abstract: Chemoenzymatic approaches using carbohydrate-active enzymes (CAZymes) offer a promising avenue for the synthesis of glycans like oligosaccharides. Here, we report a novel chemoenzymatic route for cellodextrins synthesis employed by chimeric CAZymes, akin to native glycosyltransferases, involving the unprecedented participation of a “non-catalytic” lectin-like domain or carbohydrate-binding modules (CBMs) in the catalytic step for glycosidic bond synthesis using β-cellobiosyl donor sugars as activated substrates. CBMs are often thought to play a passive substrate targeting role in enzymatic glycosylation reactions mostly via overcoming substrate diffusion limitations for tethered catalytic domains (CDs) but are not known to participate directly in any nucleophilic substitution mechanisms that impact the actual glycosyl transfer step. This study provides evidence for the direct participation of CBMs in the catalytic reaction step for β-glucan glycosidic bonds synthesis enhancing activity for CBM-based CAZyme chimeras by >140-fold over CDs alone. Dynamic intradomain interactions that facilitate this poorly understood reaction mechanism were further revealed by small-angle X-ray scattering structural analysis along with detailed mutagenesis studies to shed light on our current limited understanding of similar transglycosylation-type reaction mechanisms. In summary, our study provides a novel strategy for engineering similar CBM-based CAZyme chimeras for the synthesis of bespoke oligosaccharides using simple activated sugar monomers.

Authors: Liu, Y., Nemmaru, B., and Chundawat, S. P. S.

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Abstract: Cellulose recalcitrance toward saccharification is a barrier for low-cost biofuels production. Ammonia-based pretreatments can alter the native cellulose-I allomorphic state to form an unnatural cellulose-III allomorph that is less recalcitrant toward enzymatic hydrolysis. Here, we characterize the hydrolytic activity of a thermophilic cellulolytic microbe, Thermobifida fusca, derived cellulase on cellulose-III. Up to 2-fold improved activity was observed for homologously expressed T. Fusca cellulase enzymes on cellulose-III. Surprisingly, T. fusca exocellulases like Cel6B alone had lower activity on cellulose-III. We hypothesized that increased activity of T. fusca cellulases on cellulose-III arises mostly due to enhanced endocellulase activity and improved synergism between endo/exocellulases. Representative T. fusca endocellulase (Cel5A) and exocellulase (Cel6B) were heterologously expressed in Escherichia coli, purified, and systematically characterized for synergistic activity on cellulose-III. Hydrolytic activity assays confirmed increased activity of Cel5A on cellulose-III and improved endo/exo synergistic activity for various combinations of Cel6B/Cel5A. We finally conducted a two-step restart hydrolysis assay to also confirm if increased endoactivity results in a endo-treated cellulose-III that is amenable toward increased Cel6B activity. This work provides a mechanistic basis for increased synergistic cellulase activity on cellulose-III and provides a rationale for focusing future T. fusca enzyme engineering efforts toward potentially rate-limiting exocellulases like Cel6B.

Media Coverage: WMBCTV Interview

Authors: Chundawat, S. P. S.*, Sousa, L. daCosta, Roy, S., Yang, Z., Gupta, S., Pal, R., Zhao, C., Liu, S.-H., Petridis, L., O’Neill, H., and Pingali, S. V.

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Abstract: Here, we report novel ammonia: ammonium salt solvent-based pretreatment process that can rapidly dissolve crystalline cellulose into solution and eventually produce highly amorphous cellulose under near-ambient conditions. Pre-activating the cellulose I allomorph to its ammonia–cellulose swollen complex (or cellulose III allomorph) at ambient temperatures facilitated the rapid dissolution of the pre-activated cellulose in the ammonia-salt solvent (i.e., ammonium thiocyanate salt dissolved in liquid ammonia) at ambient pressures. For the first time in reported literature, we used time-resolved in situ neutron scattering methods to characterize the cellulose polymorphs’ structural modification and understand the mechanism of crystalline cellulose dissolution into a ‘molecular’ solution in real-time using ammonia-salt solvents. We also used molecular dynamics simulations to provide insight into solvent interactions that non-covalently disrupted the cellulose hydrogen-bonding network and understand how such solvents are able to rapidly and fully dissolve pre-activated cellulose III. Importantly, the regenerated amorphous cellulose recovered after pretreatment was shown to require nearly ∼50-fold lesser cellulolytic enzyme usage compared to native crystalline cellulose I allomorph for achieving near-complete hydrolytic conversion into soluble sugars. Lastly, we provide proof-of-concept results to further showcase how such ammonia-salt solvents can pretreat and fractionate lignocellulosic biomass like corn stover under ambient processing conditions, while selectively co-extracting ∼80–85% of total lignin, to produce a highly digestible polysaccharide-enriched feedstock for biorefinery applications. Unlike conventional ammonia-based pretreatment processes (e.g., Ammonia Fiber Expansion or Extractive Ammonia pretreatments), the proposed ammonia-salt process can operate at near-ambient conditions to greatly reduce the pressure/temperature severity necessary for conducting effective ammonia-based pretreatments on lignocellulose.

Authors: Chundawat, S. P. S.*, and Agarwal, U. P.

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Abstract: Enzymatic conversion of cellulosic biomass into fermentable sugars such as glucose is a slow and catalytically ineûcient process, largely due to limited accessibility of cellulose. Cellulose crystallinity can be reduced to increase substrate accessibility toward cellulolytic enzymes by either swelling or dissolving biomass in chemicals such as concentrated acids (e.g., phosphoric acid or H3PO4, sulfuric acid or H2SO4) or ionic liquids. Phosphoric acid swollen cellulose (PASC) is one such highly digestible form of regenerated amorphous cellulose (RAC), enriched along with some cellulose-II allomorph that can be readily produced in the lab and is, therefore, the most widely reported form of amorphous cellulose in the literature. However, concentrated hydrochloric acid (HCl) can also be used to produce RAC completely free of inorganic esters, which is structurally more representative of native cellulosic substrates and, therefore, is a useful alternative model substrate for cellulolytic enzyme assays. Here, unlike previous reports, we found that concentrated HCl swells cellulose into a gel-like state at temperatures close to freezing (4 °C), while only partially hydrolyzing and dissolving a small fraction of cellulose as large cellodextrins. Raman spectroscopy analysis suggests that cellulose-I transitions from its native allomorphic state to an intermediate swollen, gel-like state that retains some “memory” of the original starting structure, which facilitates its transition back into a cellulose-I-like state upon full solvent removal and lyophilization. Surprisingly, the cellulose regenerated from this HCl-treated cellulose gel-like state resulted in an amorphous PASC-like substrate before drying with comparable enzymatic digestibility.

Authors: Sousa, L. da C., Humpula, J., Balan, V., Dale, B. E., and Chundawat, S. P. S.

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Abstract: Plant biomasses enriched in crystalline cellulose allomorphs, such as native cellulose I (CI), can be structurally altered using anhydrous liquid ammonia to form an enzymatically less recalcitrant cellulose III (CIII) allomorph. Here, we designed and implemented an advanced ammonia pretreatment reactor/sampler setup that allowed us to systematically study the impact of several process parameters on ammonia–cellulose complex and ultimately CIII formation, including ammonia-to-cellulose concentration, ammonia/cosolvent concentration, pretreatment time, and temperature. Pretreated cellulose ultrastructural characterization was performed using complementary X-ray diffraction-, FTIR-, and FT-Raman spectroscopy-based techniques. We found that the amorphous content of cellulose increased initially when the intermediate ammonia–cellulose complex was formed within the first 30 s of pretreatment. However, a reduction in the amorphous content was observed if the complex was annealed for longer periods of time and/or at high temperatures, resulting in a highly crystalline and well-ordered CIII allomorph. However, depending on the exact pretreatment conditions tested, CIII-“like” cellulosic substrates with varying allomorphic ultrastructures and crystalline order were formed. Interestingly, CIII-like allomorphs with higher crystallinity were more easily hydrolyzed by cellulase enzymes compared to native CI and lower crystallinity CIII-like substrates. This work highlights the challenges associated with systematically conducting ammonia treatments, interpretation of the CIII ultrastructure using appropriate analytical techniques, and the influence of cellulose ultrastructure on enzymatic digestibility in light of previous work. Finally, we explored reduced severity and more cost-effective pretreatment conditions that could lower recalcitrance of cellulose by producing crystalline CIII and enable adoption of more advanced ammonia-based pretreatments in cellulosic biorefineries.