Publications

Authors: Khovesh Ramdin, Markus Hackl, Shishir PS Chundawat

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Abstract: The analysis of particles bound to a surface by flexible tethers can facilitate understanding of various biophysical phenomena (e.g., molecular dynamics of DNA-protein or protein-ligand binding interactions, DNA extensibility and polymer biophysics). Being able to model such systems theoretically can aid in understanding experimentally observed motions and furthermore the limitations of such models can provide insight into modeling complex systems that basic theory sometimes cannot account for. The simulation of tethered particle motion (TPM) allows for efficient analysis of complex behaviors exhibited by such systems, however this type of experiment is rarely taught in undergraduate science classes. We have developed a MATLAB simulation package intended to be used in academic contexts to concisely model and graphically represent the behavior of different tether-particle systems. We show how analysis of the simulation results can be used in biophysical research employing single molecule force spectroscopy (SMFS). Here, our simulation package is capable of modeling any given particle-tether-substrate system and allows the user to generate a parameter space with static and dynamic model components. Our simulation was successfully able to recreate generally observed experimental trends using a recently developed SMFS technique called Acoustic Force Spectroscopy (AFS). Further, the simulation was validated through consideration of the conservation of energy of the tether-bead system, trend analyses, and comparison of particle positional data from actual TPM in silico experiments conducted to simulate data with a parameter space similar to the AFS experimental setup. Overall, our TPM simulator and graphical user interface is suitable for use in an academic context and serves as a template for researchers to set up TPM simulations to mimic their specific SMFS experimental setup.

Authors: Aron Gyorgypal, Shishir PS Chundawat

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Abstract: The biopharmaceutical industry is transitioning toward the adoption of continuous biomanufacturing practices that are often more flexible and efficient than traditional batch processes. Federal regulatory agencies are further urging the use of advanced process analytical technology (PAT) to analyze the design space to increase the process knowledge and enable high-quality biologic production. Post-translational modifications of proteins, such as N-linked glycosylation, are often critical quality attributes that affect biologics’ safety and efficacy, requiring close monitoring during manufacturing. Here, we developed an online sequential-injection-based PAT system, called N-GLYcanyzer, which can rapidly monitor mAb glycosylation during upstream biomanufacturing. The key innovation includes the design of an integrated mAb sampling and a fully automated sample derivation system for antibody titer and glycoform.

Authors: Ayushi Agrawal, Chandra Kanth Bandi, Tucker Burgin, Youngwoo Woo, Heather B Mayes, Shishir PS Chundawat

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Abstract: Engineering of carbohydrate-active enzymes such as glycosynthases to enable chemoenzymatic synthesis of bespoke oligosaccharides has been limited by the lack of suitable ultrahigh-throughput screening methods capable of robustly detecting either starting substrates or end-products of the glycosidic bond formation reaction. Currently, there are limited screening methods available for rapid and highly sensitive single-cell-based screening of glycosynthase enzymes employing azido sugars as activated donor glycosyl substrates. Here, we report a fluorescence-based approach employing click-chemistry for the selective detection of glycosyl azides as substrates versus free inorganic azides as reaction products that facilitated an ultrahigh-throughput in vivo single-cell-based assay of glycosynthase activity.

Authors: Chandra Kanth Bandi, Kyle S Skalenko, Ayushi Agrawal, Neelan Sivaneri, Margaux Thiry, Shishir PS Chundawat

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Abstract: Detection of azide-tagged biomolecules (e.g., azido sugars) inside living cells using ‘click’ chemistry has been revolutionary to the field of chemical-biology. However, we currently still lack suitable synthetic biology tools to autonomously and rapidly detect azide ions. Here, we have developed an engineered synthetic promoter system called cyn regulon, and complementary Escherichia coli engineered strains, to selectively detect azide ions and autonomously induce downstream expression of reporter
genes. The engineered cyn azide operon allowed highly-tunable reporter green fluorescent protein (GFP) expression over three orders of inducer azide ion concentrations (0.01-5 mM) and rapidly induce GFP expression by over 600-fold compared to uninduced control. Next, we showcase the superior performance of this engineered cyn-operon over the classical lac-operon for recombinant protein production. Finally, we highlight how this synthetic biology toolkit can enable glycoengineering-based applications by facilitating in-vivo activity screening of mutant carbohydrate-active enzymes (CAZymes), called glycosynthases, using azido sugars as donor substrates.

Authors: Bhargava Nemmaru, Nicholas Ramirez, Cindy J Farino, John M Yarbrough, Nicholas Kravchenko, Shishir PS Chundawat

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Abstract: Dissociation of nonproductively 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 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 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 (𝑛𝐾_on), is correlated to overall improved endocellulase activity. Future studies could employ similar approaches to unravel the role of CBMs in non-productive enzyme binding and develop improved cellulolytic enzymes for industrial applications.

Authors: Bhargava Nemmaru, Jenna Douglass, John M Yarbrough, Antonio De Chellis, Srivatsan Shankar, Alina Thokkadam, Allan Wang, Shishir PS Chundawat

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Abstract: Non-productive adsorption of cellulolytic enzymes to various plant cell wall components, such as lignin and cellulose, necessitates high enzyme loadings to achieve efficient conversion of pretreated lignocellulosic biomass to fermentable sugars. Carbohydratebinding modules (CBMs), appended to various catalytic domains (CDs), promote lignocellulose deconstruction by increasing targeted substrate-bound CD concentration but often at the cost of increased non-productive enzyme binding. Here, we demonstrate how a computational protein design strategy can be applied to a model endocellulase enzyme (Cel5A) from Thermobifida fusca to allow fine-tuning its CBM surface charge, which led to increased hydrolytic activity towards pretreated lignocellulosic biomass (e.g., corn stover) by up to ~330% versus the wild-type Cel5A control. We established that the mechanistic basis for this improvement arises from reduced non-productive binding of supercharged Cel5A mutants to cell wall components such as crystalline cellulose (up to 1.7-fold) and lignin (up to 1.8-fold). Interestingly, supercharged Cel5A mutants that showed improved activity on various forms of pretreated corn stover showed increased reversible binding to lignin (up to 2.2-fold) while showing no change in overall thermal stability remarkably. In general, negative supercharging led to increased hydrolytic activity towards both pretreated lignocellulosic biomass and crystalline cellulose whereas positive supercharging led to a reduction of hydrolytic activity. Overall, selective supercharging of protein surfaces was shown to be an effective strategy for improving hydrolytic performance of cellulolytic enzymes for saccharification of real-world pretreated lignocellulosic biomass substrates. Future work should address the implications of supercharging cellulases from various families on inter-enzyme interactions and synergism.

Authors: Markus Hackl, Edward V Contrada, Jonathan E Ash, Atharv Kulkarni, Jinho Yoon, Hyeon-Yeol Cho, Ki-Bum Lee, John M Yarbrough, Shishir PS Chundawat

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Abstract: To rationally engineer more efficient cellulolytic enzymes for cellulosic biomass deconstruction into sugars for biofuels production, it is necessary to better understand the complex enzyme-substrate interfacial interactions. Carbohydrate binding modules (CBM) are often associated with microbial surface-tethered cellulosomal or freely secreted cellulase enzymes to increase substrate accessibility. However, it is not well known how CBM recognize, bind, and dissociate from polysaccharide surfaces to facilitate efficient cellulolytic activity due to the lack of mechanistic understanding of CBM-substrate interactions. Our work outlines a general approach to methodically study the unbinding behavior of CBMs from model polysaccharide surfaces using single-molecule force spectroscopy. Here, we apply acoustic force spectroscopy (AFS) to probe a Clostridium thermocellum cellulosomal scaffoldin protein (CBM3a) and measure its dissociation from nanocellulose surfaces at physiologically relevant, low force loading rates. An automated microfluidic setup and methodology for uniform deposition of insoluble polysaccharides on the AFS chip surfaces is demonstrated. The rupture forces of wild-type CBM3a, and its Y67A mutant, unbinding from nanocellulose surface suggests distinct CBM binding conformations that can also explain the improved cellulolytic activity of cellulase tethered to CBM. Applying established dynamic force spectroscopy theory, the single-molecule unbinding rate at zero force is extrapolated and found to agree well with bulk equilibrium unbinding rates estimated independently using quartz crystal microbalance with dissipation monitoring. However, our results highlight the limitations of applying classical theory to explain the highly multivalent CBM-cellulose interactions seen at higher cellulose-CBM bond rupture forces (>15pN).

Authors: Shishir PS Chundawat, Bhargava Nemmaru, Markus Hackl, Sonia K Brady, Mark A Hilton, Madeline M Johnson, Sungrok Chang, Matthew J Lang, Hyun Huh, Sang-Hyuk Lee, John M Yarbrough, Cesar A López, Sandrasegaram Gnanakaran

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Abstract: Efficient enzymatic saccharification of cellulosic biomass into fermentable sugars can enable production of bioproducts like ethanol. Native crystalline cellulose, or cellulose I, is inefficiently processed via enzymatic hydrolysis but can be converted into the structurally distinct cellulose III allomorph that is processed via cellulase cocktails derived from Trichoderma reesei up to 20-fold faster. However, characterization of individual cellulases from T. reesei, like the processive exocellulase Cel7A, shows reduced binding and activity at low enzyme loadings toward cellulose III. To clarify this discrepancy, we monitored the single-molecule initial binding commitment and subsequent processive motility of Cel7A enzymes and associated carbohydrate-binding modules (CBMs) on cellulose using optical tweezers force spectroscopy. We confirmed a 48% lower initial binding commitment and 32% slower processive motility of Cel7A on cellulose III, which we hypothesized derives from reduced binding affinity of the Cel7A binding domain CBM1. Classical CBM–cellulose pull-down assays, depending on the adsorption model fitted, predicted between 1.2- and 7-fold reduction in CBM1 binding affinity for cellulose III. Force spectroscopy measurements of CBM1–cellulose interactions, along with molecular dynamics simulations, indicated that previous interpretations of classical binding assay results using multisite adsorption models may have complicated analysis, and instead suggest simpler single-site models should be used. These findings were corroborated by binding analysis of other type-A CBMs (CBM2a, CBM3a, CBM5, CBM10, and CBM64) on both cellulose allomorphs. Finally, we discuss how complementary analytical tools are critical to gain insight into the complex mechanisms of insoluble polysaccharides hydrolysis by cellulolytic enzymes and associated carbohydrate-binding proteins.

Authors: Chandra Kanth Bandi, Kyle S Skalenko, Ayushi Agrawal, Neelan Sivaneri, Margaux Thiry, Shishir PS Chundawat

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Abstract: Real-time azide or azido-functionalized molecular detection inside living cells using bioorthogonal chemistry-based approaches has been revolutionary to advancing chemical-biology. These methods have enabled diverse applications ranging from understanding the role of cellular glycosylation pathways, identifying diseased cells, and targeting delivery of azido-based therapeutic drugs. However, while classical techniques were applicable only to in-vitro detection of such functional groups, even recent bioorthogonal based-detection methods require expensive sensing reagents and also cannot selectively identify inorganic azide. Here, we report an in-vivo synthetic promoter based azide biosensor toolkit to selectively detect azide anions. A promiscuous cyanate-specific promoter was engineered to detect azide and rapidly induce expression of green fluorescent protein (GFP) in Escherichia coli. Our synthetic azide operon allows highly-tunable GFP expression, outperforming the classic lac-operon, and also offers an alternative low-cost protein expression system. Finally, we showcase the utility of this toolkit for in-vivo bioorthogonal reaction biosensing and glycoengineering based applications.

Authors: Shishir PS Chundawat, Leonardo da Costa Sousa, Shyamal Roy, Zhi Yang, Shashwat Gupta, Ramendra Pal, Chao Zhao, Shih-Hsien Liu, Loukas Petridis, Hugh O’Neill, Sai Venkatesh Pingali

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Abstract: Here, we report a 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 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.