Publications

Authors: Yue Rui, Magda Zaoralová, William P Dwyer, Andres V Reyes, Tarabryn S Grismer, Nikolaj B Abel, Dharanidaran Jayachandran, Shishir PS Chundawat, Thomas Ott, Joseph J Kieber, Peter D Dahlberg, Shou-Ling Xu, José R Dinneny

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Abstract: The outer cell surface of an organism is the frontline for detecting and responding to environmental stimuli. In plants, this interface consists of the plasma membrane that lies beneath the cell wall and remains associated with it through attachment sites. These wall-membrane attachments become evident upon hyperosmotic shock, when severe water loss causes the membrane to retract from the wall. Despite their long-standing observation, the molecular identity and function of these attachments remain poorly understood. Here, we identified two mechanisms governing wall-membrane attachments: one dependent on the cellulose synthase complex (CSC), whose density at the plasma membrane positively correlates with resistance to hyperosmotic stress, and the other on remorin (REM), which acts antagonistically to the CSC mechanism. Using proximity-labeling proteomics, we identified SHOU4/4L as REM-associated proteins that mediate this antagonism. Together, our findings reveal how wall-membrane attachments are patterned to mediate plant cell resilience under water stress.

Authors: Yue Rui, Magda Zaoralová, William P. Dwyer, Andres V. Reyes, Tarabryn S. Grismer, Nikolaj B. Abel, Dharanidaran Jayachandran, Shishir P. S. Chundawat, Thomas Ott, Joseph J. Kieber, Peter D. Dahlberg, Shou-Ling Xu, José R. Dinneny

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Abstract: The outer cell surface of an organism is the frontline for detecting and responding to environmental stimuli. In plants, this interface consists of the plasma membrane that lies beneath the cell wall and remains associated with it through attachment sites. These wall-membrane attachments become evident upon hyperosmotic shock, when severe water loss causes the membrane to retract from the wall. Despite their long-standing observation, the molecular identity and function of these attachments remain poorly understood. Here, we identified two mechanisms governing wall-membrane attachments: one dependent on the Cellulose Synthase Complex (CSC), whose density at the plasma membrane positively correlates with resistance to hyperosmotic stress, and the other on REMORIN (REM), which acts antagonistically to the CSC mechanism. Using proximity-labeling proteomics, we identified SHOU4/4L as REM-associated proteins that mediate this antagonism. Together, our findings reveal how wall-membrane attachments are patterned to mediate plant cell resilience under water stress.

Authors: Shrinivas Nandi, Timothy G Stephens, Rebecca Garcia, Mayra Sánchez-García, Loretta M Roberson, Jose L Avalos, Shishir PS Chundawat, Debashish Bhattacharya

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Abstract: Massive influxes of pelagic Sargassum spp. across the tropical Atlantic and Caribbean regions have created urgent ecological and economic challenges that need to be addressed to stabilize local ecosystems. Use of this abundant biomass feedstock resource for biorefining and bioproducts manufacturing is a promising avenue, but this goal requires elucidating the microbial processes that regulate Sargassum degradation, which are still poorly understood. Here, we investigated the microbial degradation of the benthic Sargassum filipendula by native microbiota using multi-omics approaches. Metagenomic and meta-transcriptomic analyses identified diverse carbohydrate-active enzymes (CAZymes), including alginate lyases, fucoidanases, and cellulases, that were differentially expressed over the course of the in vitro degradation timeline. Furthermore, we identified the need for arsenic detoxification pathways in microbes utilizing Sargassum-derived substrates. We observed a suite of factors influencing microbial dynamics, including prokaryotic competition, arsenic detoxification, viruses, and substrate availability. Lineages potentially capable of degrading recalcitrant polysaccharides such as fucoidan appeared to be rapidly outcompeted by other bacteria that utilized simpler substrates like mannitol. These results highlight the metabolic potential of native marine microbial communities to degrade complex Sargassum polysaccharides and the importance of the in vitro degradation experiment time scale to capture the activities of non-dominant specialists. Our findings elucidate microbial ecosystem dynamics during Sargassumdegradation and provide novel insights that can be used to advance the development of biotechnological approaches that leverage renewable Sargassum biomass as a biorefinery feedstock of the future.

Authors: Antonio DeChellis, Sumay Trivedi, Lingjun Xie, Sagar Khare, Shishir PS Chundawat

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Abstract: Poly(ethylene terephthalate) (PET) is a highly recalcitrant polyester plastic whose resistance to degradation has contributed to widespread environmental accumulation. Enzymatic PET depolymerization has emerged as a promising bioremediation strategy, but PET hydrolysis remains challenging due to the insoluble and semi-crystalline nature of PET and the poor thermostability of many PET hydrolases at elevated temperatures. Here, several electrostatically supercharged PET binding modules (PBM) were fused to a PET-hydrolyzing Cutinase Catalytic Domain (CD) from the thermophilic microbe Thermobifida fusca to investigate how engineered PBM surface charge influences PET hydrolysis behavior. All PBM designs were derived from a native T. fusca family-2a carbohydrate binding module (CBM) as starting template. Since PET exhibited a substantially negative zeta potential, and accordingly, all positively supercharged PBMs displayed the strongest PET binding interactions in pull-down binding assays. However, stronger PET binding did not translate to improved hydrolysis activity for the fusion constructs. Instead, a slightly negatively charged PBM-CD fusion (D2 construct) exhibited activity comparable to the Cutinase CD on finely milled PET powder while showing substantially improved activity on intact PET discs, suggesting potential advantages for depolymerization of minimally processed PET feedstocks. Thermostability analysis identified an approximately 10 °C increase in melting temperature for the D2 fusion construct, corresponding to enhanced catalytic persistence and a shifted optimal hydrolysis temperature. Consequently, this construct exhibited an approximately 2-fold increase in long-term hydrolysis activity on milled PET and up to a 10-fold increase on intact PET discs, even at high solids loadings, compared to the native Cutinase CD. Collectively, these findings demonstrate that thermostability, rather than adsorption to PET alone, is a dominant factor governing functional persistence of PET hydrolases.

 

Authors: A. DeChellis, S. Shimabukuro, S. Trivedi, R. Lubowski, B. Nemmaru, S. P. S. Chundawat

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Abstract: Lignocellulosic biomass is an abundant renewable carbon source for biofuel production, but its conversion to fermentable sugars is hindered by poor cellulase activity on highly crystalline and insoluble cellulose. While pretreatment makes biomass more amenable to enzymatic degradation, several issues linger related to productive enzyme binding and efficient catalytic turnover. To address this bottleneck, we employed protein supercharging to rationally design a glycosyl hydrolase (GH) family-6 exocellulase (Cel6B) and its native family-2a carbohydrate binding module (CBM2a) from the thermophilic cellulolytic microbe Thermobifida fusca. A chimeric library of 32 supercharged constructs rationally designed across both GH/CBM domains was synthesized and expressed in E. coli. Screening of the entire library of supercharged enzymes on several cellulosic substrates identified one key construct, D5 CBM2a–WT Cel6B, containing a positively supercharged CBM2a that showed 2–threefold higher activity on all substrates tested at pH 5.5. Purified enzyme assays confirmed that exocellulases behave quite differently from their endocellulase counterparts when supercharged using similar protocols. Still, the purified D5 CBM2a–WT Cel6B mutant showed a 2.3-fold improvement in specific activity compared to native enzyme on crystalline cellulose. Analysis of melt curves depicts that, while all other constructs tested have one distinct melt peak near the expected CBM melting point, domain melting is decoupled for the D5 CBM2a mutant. This effect reveals an intrinsic melting temperature of the Cel6B CD nearly 18 °C higher than the coupled melting temperature of the full-length enzyme. This unexpected stabilization effect of supercharged CBM2a domain is likely the driving force for activity improvements seen for this exocellulase that is otherwise prone to stalling and denaturation on the cellulose surface during processive catalytic turnover cycles. When combining this supercharged exocellulase construct with its endocellulase counterpart, our results showed that supercharged enzymes, exhibiting the highest activity alone, produced the best synergistic partners. This study highlights another successful implementation of protein supercharging for cellulases and provides another key piece toward building an effective synergistic cellulase cocktail for lignocellulosic biomass deconstruction.

Authors: A. DeChellis, S. Shimabukuro, S. Trivedi, R. Lubowski, B. Nemmaru, S. P. S. Chundawat

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Abstract: Lignocellulosic biomass is a vastly abundant renewable carbon source for biofuel production but its conversion to fermentable sugars is significantly hindered by an inherent recalcitrance to enzymatic degradation. Pretreatment technologies are successful in alleviating some challenges related to substrate recalcitrance, yet enzymes like cellulases still exhibit poor activity on highly crystalline and insoluble cellulose. Both cellulose and lignin present several issues with productive enzyme binding and efficient catalytic turnover. To address these bottlenecks, we employed protein supercharging to rationally design a glycosyl hydrolase (GH) family-6 exocellulase (Cel6B) and its native fused family-2a carbohydrate binding module (CBM2a) from the thermophilic cellulolytic microbe Thermobifida fusca. A total of 16 supercharged variants were designed across both GH/CBM domains and a chimeric library of 32 constructs, including the native enzyme, were synthesized and expressed in E. coli. The entire library of supercharged enzymes was tested for activity on several cellulosic substrates to identify one key construct, D5 CBM2a – WT Cel6B, that had a positively supercharged CBM2a that showed 2-3-fold higher activity on all substrates tested at pH 5.5. Purified enzyme assays confirmed that exocellulases behave quite different from their endocellulase counterparts when supercharged using similar protocols. Still, the purified D5 CBM2a – WT Cel6B mutant showed a 2.3-fold improvement in specific activity compared to native enzyme on crystalline cellulose. Analysis of melt curves depict that, while all other constructs tested have one distinct melt peak near the expected CBM melting point, domain melting is decoupled for the D5 CBM2a mutant. This effect reveals an intrinsic melting temperature of the Cel6B CD nearly 18 °C higher than the coupled melting temperature of the full-length enzyme. This unexpected catalytic domain stabilization effect of supercharged CBM2a domain is likely the driving force for activity improvements seen for this exocellulase that is otherwise prone to stalling and denaturation on the cellulose surface during processive catalytic turnover cycles. When combining this supercharged exocellulase construct with its endocellulase counterpart, our results show that supercharged enzymes that show the highest activity alone, produced the best synergistic partners. This study highlights another successful implementation of protein supercharging strategy for cellulases and provides another key piece towards building an effective synergistic cellulase cocktail for lignocellulosic biomass deconstruction.

 

Authors: Nurulhuda Binte Suaini, Aditya Narvekar, Shishir PS Chundawat

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Abstract: N-glycosylation is a post-translational modification of proteins that represents a critical quality attribute (CQA) for therapeutics like monoclonal antibodies (mAbs), directly affecting drug efficacy, safety, and stability. Real-time CQA monitoring analytical platforms depend on rapid N-glycan release and fluorophore labeling chemistries to support automated bioprocess analytics during mAb manufacturing. Procainamide is a well-known fluorophore used for released N-glycans reducing sugar aldehydes labeling that offers both high fluorescence and mass spectrometry detection sensitivity comparable to several commercial reagents available in the market. However, currently there are no studies that optimize its use and long incubation times are often reported in the literature for procainamide labeling of N-glycans that has limited its use in time-sensitive workflows relevant to various stakeholders in industry, academia, and regulatory agencies. Here, we have systematically determined the combined procainamide labeling via reductive amination/reduction reaction kinetics at various incubation intervals, ranging from 1 min to 12 h, using N-glycans isolated from model biologic glycoprotein trastuzumab (TmAb). Labeling efficiencies were quantified using high-performance liquid chromatography with fluorescence detection (HPLC-FLR), and detailed reaction parameters were determined by fitting suitable kinetic models. Results indicate that most N-glycans reached over 95% labeling efficiency within 1 hour at the desired reaction temperature. Interestingly, N-glycan structural features, particularly galactosylation and fucosylation levels, significantly influenced the labeling reaction rate. Fucosylated glycans exhibited up to 4-fold higher reaction rate constants than non-fucosylated forms, whereas increased galactosylation levels was associated with slower reaction rate. These results provide essential kinetic benchmarks for incorporating procainamide labeling for released N-glycans, and facilitating more efficient analytical workflows for Process Analytical Technology (PAT) focused on biologics N-glycan analysis in both research and industrial settings.

Authors: Aron Gyorgypal, Erica Fratz‐Berilla, Casey Kohnhorst, David N Powers, Shishir PS Chundawat

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Abstract: Monoclonal antibodies (mAbs) represent a majority of biotherapeutics in the market today. These glycoproteins undergo posttranslational modifications, such as N-linked glycosylation, that influence the structural & functional characteristics of the antibody. Glycosylation is a heterogenous posttranslational modification that may influence therapeutic glycoprotein stability and clinical efficacy, which is why it is often considered a critical quality attribute (CQA) of the mAb product. While much is known about the glycosylation pathways of Chinese Hamster Ovary (CHO) cells and how cell culture chemical modifiers may influence the N-glycosylation profile of the final product, this knowledge is often based on the final cumulative glycan profile at the end of the batch process. Building a temporal understanding of N-glycosylation and how mAb glycoform composition responds to real-time changes in the biomanufacturing process will help build integrated process models that may allow for glycosylation control to produce a more homogenous product. Here, we look at the effect of specific nutrient feed media additives (e.g., galactose, manganese) and feeding times on the N-glycosylation pathway to modulate N-glycosylation of a Herceptin biosimilar mAb (i.e., Trastuzumab). We deploy the N-GLYcanyzer process analytical technology (PAT) to monitor glycoforms in near real-time for bench-scale bioprocesses operated in both fed-batch and perfusion modes to build an understanding of how temporal changes in mAb N-glycosylation are dependent on specific media additives. We find that Trastuzumab terminal galactosylation is sensitive to media feeding times and intracellular nucleotide sugar pools. Temporal analysis reveals an increased desirable production of single and double galactose-occupied glycoforms over time under glucose-starved fed-batch cultures. Comparable galactosylation profiles were also observed between fed-batch (nutrient-limited) and perfusion (non-nutrient-limited) bioprocess conditions. In summary, our results demonstrate the utility of real-time monitoring of mAb glycoforms and feeding critical cell culture nutrients under fed-batch and perfusion bioprocessing conditions to produce higher-quality biologics.

Authors: Hyun Huh, Dharanidaran Jayachandran, Junhong Sun, Mohammad Irfan, Eric Lam, Shishir PS Chundawat, Sang-Hyuk Lee

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Abstract: Cellulose, the most abundant polysaccharide on earth composing plant cell walls, is synthesized by coordinated action of multiple enzymes in cellulose synthase complexes embedded within the plasma membrane. Multiple chains of cellulose fibrils form intertwined extracellular matrix networks. It remains largely unknown how newly synthesized cellulose is assembled into an intricate fibril network on cell surfaces. Here, we have established an in vivo time-resolved imaging platform to continuously visualize cellulose biosynthesis and fibril network assembly on Arabidopsis thaliana protoplast surfaces as the primary cell wall regenerates. Our observations provide the basis for a model of cellulose fibril network development in protoplasts driven by an interplay of multiscale dynamics that includes rapid diffusion and coalescence of nascent cellulose fibrils, processive elongation of single fibrils, and cellulose fibrillar network rearrangement during maturation. This study provides fresh insights into the dynamic and mechanistic aspects of cell wall synthesis at the single-cell level.

Authors: Aron Gyorgypal, Antash Chaturvedi, Viki Chopda, Haoran Zhang, Shishir PS Chundawat

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Abstract:

The choice of media and feeds significantly influences the performance of Chinese Hamster Ovary (CHO) mammalian cell cultures in producing desired biologics like monoclonal antibodies (mAb). Sub-optimal nutrient feed/media composition can severely impact cell proliferation and the quality of the final mAb product. For instance, proper protein glycosylation, crucial for mAb stability, safety, and efficacy, heavily relies on cell culture conditions. Currently, starter CHO culture media and daily supplemental feeds used in industrial manufacturing consist of proprietary composition of nutrients critical for mAb production. Standardized optimal media/feed combinations necessary for different cell lines are often lacking, necessitating individualized optimization for each cell line and mAb product. Here, we focused on a CHO-K1 cell line engineered to produce a Trastuzumab biosimilar and evaluated the effects of fourteen commercially relevant basal media and seven feeds on cell culture parameters such as viable cell density, viability, nutrient consumption, metabolite production, mAb titer, and mAb N-glycosylation. Our findings demonstrate clearly that the compositions of the basal medium and feed play a pivotal role in enhancing cell growth and mAb production. This work offers valuable insights into strategies for optimizing feed/media composition for glycosylated monoclonal antibody production using CHO cells.

Authors: Ashley Dan, Bochi Liu, Urjit Patil, Bhavani Nandhini Mummidi Manuraj, Ronit Gandhi, Justin Buchel, Shishir PS Chundawat, Weihong Guo, Rohit Ramachandran

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Abstract: This study is concerned with the development of reduced order machine learning (ML) and non-ML model representations of experimental and simulated bioprocesses and their implementation in model predictive control (MPC) strategies to quantify performance accuracy and computational efficiency compared with the original models. Results showed that ML models such as Long Short-Term Memory (LSTM) networks and Artificial Neural Networks (ANNs) outperformed other reduced order models such as Kriging, Multiple Linear Regression (MLR) and Random Forest (RF) in terms of performance metrics such as R² and RMSE for both experimental and simulated data. Experimental data were obtained from a fed-batch and perfusion-based bioprocess and an LSTM model was developed and implemented in an MPC open-loop optimal control strategy to determine optimal input trajectories to maximize key performance metrics such as product titer. For the 2 by 3 ODE simulation, results showed that an autoregressive ANN was the most accurate in terms of replicating the plant model dynamics under MPC conditions followed by the LSTM and transfer function (TF) representations, with the feedforward ANN not being able to fully capture the salient dynamics. For the 4 by 5 ODE simulation, the TF representation outperformed the feedforward ANN model which in turn was more accurate than the LSTM model. In terms of computational time, the plant model simulation time for an MPC solution is intractable for larger input-output sizes compared with the ML models. Overall, it can be seen the ML models such as ANNs and LSTMs, provide the best balance between accuracy and computational efficiency as they can capture the inherent nonlinearities of the plant model but also are not computationally intensive compared to the full plant model which are often represented by ODE and/or PDE-based differential equations. ML models such as those developed in this study demonstrate practical methods of implementing advanced process control in highly nonlinear chemical/biological processes as part of the smart manufacturing/Industry 4.0 paradigm.

Authors: Madeline M Johnson, Antonio DeChellis, Bhargava Nemmaru, Shishir PS Chundawat, Matthew J Lang

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Abstract: Cellulose, an abundant biopolymer, has great potential to be utilized as a renewable fuel feedstock through its enzymatic degradation into soluble sugars followed by sugar fermentation into liquid biofuels. However, crystalline cellulose is highly resistant to hydrolysis, thus industrial-scale production of cellulosic biofuels has been cost-prohibitive to date. Mechanistic studies of enzymes that break down cellulose, called cellulases, are necessary to improve and adapt such biocatalysts for implementation in biofuel production processes. Thermobifida fusca Cel6B (TfCel6B) is a promising candidate for industrial use due to its thermostability and insensitivity to pH changes. However, mechanistic studies probing TfCel6B hydrolytic activity have been limited to ensemble-scale measurements. We utilized optical tweezers to perform single-molecule, nanometer-scale measurements of enzyme displacement during cellulose hydrolysis by TfCel6B. Records featured forward motility on the order of 0.17 nm s⁻¹ interrupted by backward motions and long pauses. Processive run lengths were on the order of 5 nm in both forward and backward directions. Motility records also showed rapid bidirectional displacements greater than 5 nm. Single-enzyme velocity and bulk ensemble activity were assayed on multiple crystalline cellulose allomorphs revealing that the degree of crystallinity and hydrogen bonding have disparate effects on the single-molecule level compared to the bulk scale. Additionally, we isolated and monitored the catalytic domain of TfCel6B and observed a reduction in velocity compared to the full-length enzyme that includes the carbohydrate-binding module. Applied force has little impact on enzyme velocity yet it readily facilitates dissociation from cellulose. Preliminary measurements at elevated temperatures indicated enzyme velocity strongly increases with temperature. The unexpected motility patterns of TfCel6B are likely due to previously unknown mechanisms of processive cellulase motility implicating irregularities in cellulose substrate ultrastructure. While TfCel6B is processive, it has low motility at room temperature. Factors that most dramatically impact enzyme velocity are temperature and the presence of its native carbohydrate-binding module and linker. In contrast, substrate ultrastructure and applied force did not greatly impact velocity. These findings motivate further study of TfCel6B for its engineering and potential implementation in industrial processes.

Authors: Hyun Huh, Dharanidaran Jayachandran, Junhong Sun, Mohammad Irfan, Eric Lam, Shishir PS Chundawat, Sang-Hyuk Lee

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Abstract: Plant cell walls are composed of polysaccharides among which cellulose is the most abundant component. Cellulose is processively synthesized as bundles of linear β-1,4-glucan homopolymer chains via the coordinated action of multiple enzymes in cellulose synthase complexes (CSCs) embedded within the plasma cell membrane. Plant cell walls are composed of multiple layers of cellulose fibrils that form highly intertwined extracellular matrix networks. However, it is not yet clear as to how cellulose fibrils synthesized by multiple CSCs are assembled into the intricate cellulose network deposited on plant cell surfaces. Herein, we have established an in vivo time-resolved imaging platform for visualizing cellulose during its biosynthesis and assembly into a complex fibrillar network on the surface of Arabidopsis thaliana mesophyll protoplasts as the primary cell wall regenerates. We performed total internal reflection fluorescence microscopy (TIRFM) with fluorophore-conjugated tandem carbohydrate binding modules (tdCBMs) that were engineered to specifically bind to nascent cellulose fibrils. Together with a well-controlled environment, it was possible to monitor in vivocellulose fibril synthesis dynamics in a time-resolved manner for nearly one day of continuous cell wall regeneration on protoplast cell surfaces. Our observations provide the basis for a novel model of cellulose fibril network development in protoplasts driven by complex interplay of multi-scale dynamics that include: rapid diffusion and coalescence of short nascently synthesized cellulose fibrils; processive elongation of single fibrils; and cellulose fibrillar network rearrangement during cell wall maturation. This platform is valuable for exploring mechanistic aspects of cell wall synthesis while visualizing cellulose microfibrils assembly.

Significance Statement Cellulose is a major extracellular matrix component of cells that is critical for plant development and has applications to bioenergy, agricultural food/feed, textile, and wood production. Cellulose is thought to be assembled by the closely coordinated motion of plasma membrane-embedded cellulose synthase enzyme complexes. To date, however, it has not been possible to visualize de novo plant cell wall synthesis at the single cell level with the necessary spatiotemporal resolution to derive a data-driven model of how plant cells can resynthesize and assemble cell wall after its removal. Based on our time-resolved data, we propose a new model for cellulose biosynthesis after successfully performing live protoplast time-lapse imaging to visualize for the first time the complex dynamics of de novo cellulose biosynthesis and assembly into an intertwined microfibril network.

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

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Abstract: Non-productive binding 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. Protein supercharging was previously employed as one of the strategies to reduce non-productive binding to biomass. However, various questions remain unanswered regarding the hydrolysis kinetics of supercharged enzymes towards pretreated biomass substrates and the role played by enzyme interactions with individual cell wall polymers such as cellulose and xylan. In this study, CBM2a (from Thermobifida fusca) fused with endocellulase Cel5A (from T. fusca) was used as the model wild-type enzyme and CBM2a was supercharged using Rosetta, to obtain eight variants with net charges spanning −14 to +6. These enzymes were recombinantly expressed in E. coli, purified from cell lysates, and their hydrolytic activities were tested against pretreated biomass substrates (AFEX and EA treated corn stover). Although the wild-type enzyme showed greater activity compared to both negatively and positively supercharged enzymes towards pretreated biomass, thermal denaturation assays identified two negatively supercharged constructs that perform better than the wild-type enzyme (∼3 to 4-fold difference in activity) upon thermal deactivation at higher temperatures. To better understand the causal factor of reduced supercharged enzyme activity towards AFEX corn stover, we performed hydrolysis assays on cellulose-I/xylan/pNPC, lignin inhibition assays, and thermal stability assays. Altogether, these assays showed that the negatively supercharged mutants were highly impacted by reduced activity towards xylan whereas the positively supercharged mutants showed dramatically reduced activity towards cellulose and xylan. It was identified that a combination of impaired cellulose binding and lower thermal stability was the cause of reduced hydrolytic activity of positively supercharged enzyme sub-group. Overall, this study demonstrated a systematic approach to investigate the behavior of supercharged enzymes and identified supercharged enzyme constructs that show superior activity at elevated temperatures. Future work will address the impact of parameters such as pH, salt concentration, and assay temperature on the hydrolytic activity and thermal stability of supercharged enzymes.

Authors: Dharanidaran Jayachandran, Amar Parvate, Jory T Brookreson, James E Evans, Shishir PS Chundawat

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Abstract: Polysaccharides are a major class of natural polymers found abundantly across all major life forms and play a critical role as structural, metabolic, or functional components in biomolecular processes. Some polysaccharides like cellulose and hyaluronan are synthesized by membrane-bound family-2 glycosyltransferases (GTs). Despite the fact that the GT-2 family has the maximum number of deposited sequences, the biochemistry of GT-2 family enzymes is still poorly understood due to difficulties associated with GT membrane protein expression, purification, and reconstitution in lipid carriers. Here, we chose Populus tremula x tremuloides cellulose synthase 8 (PttCesA8) and Streptococcus equisimilishyaluronan synthase (SeHas) as putative family-2-GTs to be expressed in a wheat-germ-based cell-free expression (CFE) system as proteoliposomes. The cell-free products were obtained as reconstituted liposomes directly from CFE reactions at high yields and short processing times compared to other approaches. GT enzymes expression was confirmed using SDS-PAGE and immunoblotting, and the integration of GTs in lipid layers was observed using cryogenic electron microscopy. Both GTs tested were catalytically active when incubated with their respective substrates and cofactors. The Michalis-Menten kinetic constants, K_m for PttCesA8, was 295.8 µM, and SeHas was 321.51 µM (toward UDP N-Acetyl Glucosamine) and 207.88 µM (toward UDP Glucuronic Acid), respectively. UDP was found to actively inhibit both these GTs with apparent inhibition constants of 10.08 µM and 24.38 µM. Mutation of specific conserved residues in structure-deficit SeHas confirmed the importance of lysine-139, glutamine-248, and threonine-283 residues in hyaluronan biosynthesis. In summary, wheat-germ-based CFE can be used to express functionally active and liposome-reconstituted family-2 GTs at high yields with relative ease to enable classical enzymology assays and will also enable more detailed structural studies in the near future.

Authors: Antonio DeChellis, Bhargava Nemmaru, Deanne Sammond, Jenna Douglass, Nivedita Patil, Olivia Reste, Shishir PS Chundawat

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Abstract: Lignocellulosic biomass recalcitrance to enzymatic degradation necessitates high enzyme loadings, incurring large processing costs for the production of industrial-scale biofuels or biochemicals. Manipulating surface charge interactions to minimize nonproductive interactions between cellulolytic enzymes and plant cell wall components (e.g., lignin or cellulose) via protein supercharging has been hypothesized to improve biomass biodegradability but with limited demonstrated success to date. Here, we characterize the effect of introducing non-natural enzyme surface mutations and net charge on cellulosic biomass hydrolysis activity by designing a library of supercharged family-5 endoglucanase Cel5A and its native family-2a carbohydrate binding module (CBM) originally belonging to an industrially relevant thermophilic microbe, Thermobifida fusca. A combinatorial library of 33 mutant constructs containing different CBM and Cel5A designs spanning a net charge range of −52 to 37 was computationally designed using Rosetta macromolecular modeling software. Activity for all mutants was rapidly characterized as soluble cell lysates, and promising mutants (containing mutations on the CBM, Cel5A catalytic domain, or both CBM and Cel5A domains) were then purified and systematically characterized. Surprisingly, often endocellulases with mutations on the CBM domain alone resulted in improved activity on cellulosic biomass, with three top-performing supercharged CBM mutants exhibiting between 2- and 5-fold increase in activity, compared to native enzyme, on both pretreated biomass enriched in lignin (i.e., corn stover) and isolated crystalline/amorphous cellulose. Furthermore, we were able to clearly demonstrate that endocellulase net charge can be selectively fine-tuned using a protein supercharging protocol for targeting distinct substrates and maximizing biocatalytic activity. Additionally, several supercharged CBM-containing endocellulases exhibited a 5–10 °C increase in optimal hydrolysis temperature, compared to native enzyme, which enabled further increase in hydrolytic yield at higher operational reaction temperatures. This study demonstrates the first successful implementation of enzyme supercharging of cellulolytic enzymes to increase hydrolytic activity toward complex lignocellulosic biomass-derived substrates.

Authors: Khovesh Ramdin, Markus Hackl, Shishir PS Chundawat

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Abstract: The analysis of particles bound to surfaces by tethers can facilitate understanding of biophysical phenomena (e.g., DNA–protein or protein–ligand interactions and DNA extensibility). Modeling such systems theoretically aids in understanding experimentally observed motions, and the limitations of such models can provide insight into modeling complex systems. The simulation of tethered particle motion (TPM) allows for 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 using single-molecule force spectroscopy (SMFS). Students in physics, engineering, and chemistry will be able to make connections with principles embedded in the field of study and understand how those principles can be used to create meaningful conclusions in a multidisciplinary context. The simulation package can model any given tether–particle 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 by using 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 primarily for demonstrating behaviors characteristic to TPM in a classroom setting but can serve as a template for researchers to set up TPM simulations to mimic a specific SMFS experimental setup.