Publications
Dr. Chundawat’s complete list of publications is available here and at . Current h-index is 39, i10-index is 72, and total citations are 8284 (updated 2024/01). Corresponding author/s highlighted by an asterisk (*). Peer-reviewed papers are available on the publisher’s website, RUcore, and some older papers are also posted on Dr. Chundawat’s personal ResearchGate account. Original preprints are available on the bioRxiv and chemRxiv websites. Patents are available on Google Patents website.
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Authors: Whitehead TA*, Bandi CK, Berger M, Park J, Chundawat SPS
Media Coverage: Journal Cover Article Receives Extensive Media Coverage
Paper Link: Link
Journal Link: Link
Abstract: Nonspecific adsorption of cellulases to lignin hinders enzymatic biomass deconstruction. Here, we tested the hypothesis that negatively supercharging cellulases could reduce lignin inhibition. The computational design was used to negatively supercharge the surfaces of Ruminoclostridiumthermocellum family 5 CelE and a CelE-family 3a carbohydrate-binding module fusion. The resulting designs maintained the same expression yield, thermal stability, and nearly identical activity on the soluble substrates as the wild-type proteins. Four designs showed a complete lack of inhibition by lignin but with lower cellulose conversion compared to original enzymes. Increasing salt concentrations could partially rescue the activity of supercharged enzymes, supporting a mechanism of electrostatic repulsion between designs and cellulose. Results showcase a protein engineering strategy to construct highly active cellulases that are resistant to lignin-mediated inactivation, although further work is needed to understand the relationship between negative protein surface potential and activity on insoluble polysaccharides.
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Authors: Chundawat SPS*, Uppugundla N, Gao D, Curran P, Balan V, and Dale B
Journal Link: Link
Abstract: Most cellulolytic enzyme blends, either procured from a commercial vendor or isolated from a single cellulolytic microbial secretome, do not efficiently hydrolyze ammonia-pretreated (e.g., ammonia fiber expansion, AFEX) lignocellulosic agricultural crop residues like corn stover to fermentable sugars. Typically reported commercial enzyme loading (30–100 mg protein/g glucan) necessary to achieve >90% total hydrolysis yield (to monosaccharides) for AFEX-treated biomass, within a short saccharification time frame (24–48 h), is economically unviable. Unlike acid-based pretreatments, AFEX retains most of the hemicelluloses in the biomass and therefore requires a more complex suite of enzymes for efficient hydrolysis of cellulose and hemicellulose at industrially relevant high solids loadings. One strategy to reduce enzyme dosage while improving cocktail effectiveness for AFEX-treated biomass has been to use individually purified enzymes to determine optimal enzyme combinations to maximize hydrolysis yields. However, this approach is limited by the selection of heterologous enzymes available or the labor required for isolating low-abundance enzymes directly from the microbial secretomes. Here, we show that directly blending crude cellulolytic and hemicellulolytic enzymes-rich microbial secretomes can maximize specific activity on AFEX-treated biomass without having to isolate individual enzymes. Fourteen commercially available cellulolytic and hemicellulolytic enzymes were procured from leading enzyme companies (Novozymes®, Genencor®, and Biocatalysts®) and were mixed together to generate several hundred unique cocktail combinations. The mixtures were assayed for activity on AFEX-treated corn stover (AFEX-CS) using a previously established high-throughput methodology. The optimal enzyme blend combinations identified from these screening assays were enriched in various low-abundance hemicellulases and accessory enzymes typically absent in most commercial cellulases cocktails. Our simple approach of blending crude commercially available enzyme cocktails allowed a drastic fourfold reduction in total enzyme requirements (from 30 to 7.5 mg enzyme/g glucan loading) to achieve near-theoretical cellulose and hemicellulose saccharification yields for AFEX-CS.
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Authors: Haarmeyer CN, Smith MD, Chundawat SPS, Sammond D, Whitehead TA*
Paper Link: Link
Journal Link: Link
Abstract: Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue towards energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor driving cost is cellulase adsorption and inactivation in the presence of lignin, yet we currently have a poor understanding of the protein structure-function relationships driving this adsorption. In this work, we have systematically investigated the role of protein surface potential on lignin adsorption using a model monomeric fluorescent protein. We have designed and experimentally characterized 16 model protein variants spanning the physiological range of net charge (-24 to +16 total charges) and total charge density (0.28 to 0.40 charges per sequence length) typical for natural proteins. Protein designs were expressed, purified, and subjected to in silico and in vitro biophysical measurements to evaluate the relationship between protein surface potential and lignin adsorption properties. The designs were comparable to model fluorescent protein in terms of thermostability and heterologous expression yield, although the majority of the designs unexpectedly formed homodimers. Protein adsorption to lignin was studied at two different temperatures using Quartz Crystal Microbalance with Dissipation Monitoring and a subtractive mass balance assay. We found a weak correlation between protein net charge and protein-binding capacity to lignin. No other single characteristic, including apparent melting temperature and 2nd virial coefficient, showed a correlation with lignin binding. Analysis of an unrelated cellulase dataset with mutations localized to a family I carbohydrate-binding module showed a similar correlation between net charge and lignin binding capacity. Overall, our study provides strategies to identify highly active, low lignin-binding cellulases by either rational design or by computational screening genomic databases.
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Authors: Chundawat SPS*, Paavola CD, Raman B, Nouailler M, Chan SL, Mielenz JR, Brechot VR, Trent JD, Dale BE
Media Coverage: Research on engineering cellulolytic enzyme complexes for biomass deconstruction highlighted as RSC Journal Cover Article
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Abstract: Consolidated bioprocessing (CBP) of pretreated lignocellulosic biomass using microbes like Clostridium thermocellum allows simultaneous polysaccharide saccharification and sugar fermentation to produce fuels or chemicals using a one-pot process. C. thermocellum is a thermophilic bacterium that deconstructs biomass using large multi-enzyme complexes called cellulosomes. Characterization of cellulosomal enzymes tethered to native or engineered scaffoldin proteins has revealed that enzyme complexation is critical to the bacterium’s cellulolytic ability. However, we have a limited understanding of the impact of enzyme complexation on the saccharification efficiency of various forms of industrially relevant pretreated biomass substrates. Here, we compared the hydrolytic activity of the most abundant cellulosomal enzymes from C. thermocellum and investigate the importance of enzyme complexation using a model engineered protein scaffold (called ‘rosettasome’). The hydrolytic performance of non-complexed enzymes, enzyme-rosettasome (or rosettazyme) complexes, and cellulosomes was tested on distinct cellulose allomorphs formed during biomass pretreatment. The scaffold-immobilized enzymes always gave higher activity than free enzymes. However, cellulosomes exhibited higher activity than rosettazyme complexes. This was likely due to the greater flexibility of the native versus engineered scaffold, as deciphered using small angle X-ray scattering. Surprisingly, scaffold-tethered enzymes also gave comparable activity on all the cellulose allomorphs tested, which is unlike the preferential activity of non-complexed cellulases seen for certain allomorph forms. Tethered enzyme complexes also gave lower saccharification yields on industrially relevant lignin-rich switchgrass than cellulose alone. In summary, we find that the type of pretreatment can significantly impact the saccharification efficiency of cellulosomal enzymes for various CBP scenarios.
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Authors: Sousa L*, Bals B, Jin M, Chundawat SPS, Bokade V, Tang X, Azarpira A, Lu F, Avci U, Humpula J, Uppugundla N, Gunawan C, Pattathil S, Cheh A, Kothari N, Kumar R, Ralph J, Hahn MG, Wyman CE, Singh S, Simmons BA, Dale BE*, Balan V*
Media Coverage: Next Generation Pretreatments for Cellulosic Biofuels
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Abstract: A new liquid ammonia pretreatment methodology called Extractive Ammonia (EA) was developed to simultaneously convert native crystalline cellulose Iβ (CI) to a highly digestible cellulose IIII (CIII) allomorph and selectively extract up to ∼45% of the lignin from lignocellulosic biomass with near-quantitative retention of all polysaccharides. EA pretreated corn stover yielded a higher fermentable sugar yield compared to the older Ammonia Fiber Expansion (AFEX) process while using 60% lower enzyme loading. The EA process preserves extracted lignin functionalities, offering the potential to co-produce lignin-derived fuels and chemicals in the biorefinery. The single-stage EA fractionation process achieves high biofuel yields (18.2 kg ethanol per 100 kg untreated corn stover, dry weight basis), comparable to those achieved using ionic liquid pretreatments. The EA process achieves these ethanol yields at industrially-relevant conditions using low enzyme loading (7.5 mg protein per g glucan) and high solids loading (8% glucan, w/v).
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Authors: Brady S, Sarangapani S, Feng Y, Chundawat SPS, Lang MJ*
Media Coverage: Anatomy of a Microscopic Wood Chipper
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Abstract: Cellobiohydrolase 1 from Trichoderma reesei (TrCel7A) processively hydrolyses cellulose into cellobiose. Although enzymatic techniques have been established as promising tools in biofuel production, a clear understanding of the motor’s mechanistic action has yet to be revealed. Here, we develop an optical tweezers-based single-molecule (SM) motility assay for precision tracking of TrCel7A. Direct observation of motility during degradation reveals processive runs and distinct steps on the scale of 1 nm. Our studies suggest TrCel7A is not mechanically limited, can work against 20 pN loads and speeds up when assisted. Temperature-dependent kinetic studies establish the energy requirements for the fundamental stepping cycle, which likely includes energy from glycosidic bonds and other sources. Through SM measurements of isolated TrCel7A domains, we determine that the catalytic domain alone is sufficient for processive motion, providing insight into TrCel7A’s molecular motility mechanism.
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Authors: O’Neill H*, Shah R, Evans BR, He J, Pingali SV, Chundawat SPS, Jones AD, Langan P, Davison BH, Urban V.
Journal Link: Link
Abstract: Isotopic enrichment of biomacromolecules is a widely used technique that enables the investigation of the structural and dynamic properties to provide information not accessible with natural abundance isotopic composition. This study reports an approach for deuterium incorporation into bacterial cellulose. A media formulation for growth of Acetobacter xylinus subsp. sucrofermentans and Gluconacetobacter hansenii was formulated that supports cellulose production in deuterium (D) oxide. The level of D incorporation can be varied by altering the ratio of deuterated and protiated glycerol used during cell growth in the D2O-based growth medium. Spectroscopic analysis and mass spectrometry show that the level of deuterium incorporation is high (>90%) for the perdeuterated form of bacterial cellulose. The small-angle neutron scattering profiles of the cellulose with different amounts of D incorporation are all similar indicating that there are no structural changes in the cellulose due to substitution of deuterium for hydrogen. In addition, by varying the amount of deuterated glycerol in the media it was possible to vary the scattering length density of the deuterated cellulose. The ability to control deuterium content of cellulose extends the range of experiments using techniques such as neutron scattering to reveal information about the structure and dynamics of cellulose, and its interactions with other biomacromolecules as well as synthetic polymers used for development of composite materials.
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Authors: Pattathil S*, Hahn M, Dale BE, Chundawat SPS*
Journal Link: Link
Abstract: Cell walls, which constitute the bulk of plant biomass, vary considerably in their structure, composition, and architecture. Studies on plant cell walls can be conducted on both native and pre-treated plant biomass samples, allowing an enhanced understanding of these structural and compositional variations. Here glycome profiling was employed to determine the relative abundance of matrix polysaccharides in several phylogenetically distinct native and pre-treated plant biomasses. Eight distinct biomass types belonging to four different subgroups (i.e. monocot grasses, woody dicots, herbaceous dicots, and softwoods) were subjected to various regimes of AFEX™ (ammonia fiber expansion) pre-treatment [AFEX is a trademark of MBI, Lansing (http://www.mbi.org]. This approach allowed detailed analysis of close to 200 cell wall glycan epitopes and their relative extractability using a high-throughput platform. In general, irrespective of the phylogenetic origin, AFEX™ pre-treatment appeared to cause loosening and improved accessibility of various xylan epitope subclasses in most plant biomass materials studied. For most biomass types analysed, such loosening was also evident for other major non-cellulosic components including subclasses of pectin and xyloglucan epitopes. The studies also demonstrate that AFEX™ pre-treatment significantly reduced cell wall recalcitrance among diverse phylogenies (except softwoods) by inducing structural modifications to polysaccharides that were not detectable by conventional gross composition analyses. It was found that monitoring changes in cell wall glycan compositions and their relative extractability for untreated and pre-treated plant biomass can provide an improved understanding of variations in structure and composition of plant cell walls and delineate the role(s) of matrix polysaccharides in cell wall recalcitrance.
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Authors: López C, Bellesia G, Redondo A, Langan P, Chundawat SPS, Dale BE, Marrink S, Gnanakaran S*
Paper Link: Link
Journal Link: Link
Abstract: Commercial-scale biofuel production requires a deep understanding of the structure and dynamics of its principal target: cellulose. However, an accurate description and modeling of this carbohydrate structure at mesoscale remains elusive, particularly because of its overwhelming length scale and configurational complexity. We have derived a set of MARTINI coarse-grained force field parameters for the simulation of crystalline cellulose fibers. The model is adapted to reproduce different physicochemical and mechanical properties of native cellulose Iβ. The model is not only able to handle a transition from cellulose Iβ to another cellulose allomorph, cellulose IIIi, but to capture the physical response to temperature and mechanical bending of longer cellulose nano- fibers. By developing the MARTINI model of a solid cellulose crystalline fiber from the building blocks of a soluble cellobiose coarse-grained model, we have provided a systematic way to build MARTINI models for other crystalline biopolymers.
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Authors: Tang X, Sousa L, Jin M, Chundawat SPS, Chambliss CK, Lau MW, Xiao Z, Dale BE, Balan V
Paper Link: Link
Journal Link: Link
Abstract: The fermentation inhibition of yeast or bacteria by lignocellulose-derived degradation products, during hexose/pentose co-fermentation, is a major bottleneck for cost-effective lignocellulosic biorefineries. To engineer microbial strains for improved performance, it is critical to understand the mechanisms of inhibition that affect fermentative organisms in the presence of major components of a lignocellulosic hydrolysate. The development of a synthetic lignocellulosic hydrolysate (SH) media with a composition similar to the actual biomass hydrolysate will be an important advancement to facilitate these studies. In this work, we characterized the nutrients and plant-derived decomposition products present in AFEX™ pretreated corn stover hydrolysate (ACH). The SH was formulated based on the ACH composition and was further used to evaluate the inhibitory effects of various families of decomposition products during Saccharomyces cerevisiae 424A (LNH-ST) fermentation. RESULTS: The ACH contained high levels of nitrogenous compounds, notably amides, pyrazines, and imidazoles. In contrast, a relatively low content of furans and aromatic and aliphatic acids were found in the ACH. Though most of the families of decomposition products were inhibitory to xylose fermentation, due to their abundance, the nitrogenous compounds showed the most inhibition. From these compounds, amides (products of the ammonolysis reaction) contributed the most to the reduction of the fermentation performance. However, this result is associated to a concentration effect, as the corresponding carboxylic acids (products of hydrolysis) promoted greater inhibition when present at the same molar concentration as the amides. Due to its complexity, the formulated SH did not perfectly match the fermentation profile of the actual hydrolysate, especially the growth curve. However, the SH formulation was effective for studying the inhibitory effect of various compounds on yeast fermentation. CONCLUSIONS: The formulation of SHs is an important advancement for future multi-omics studies and for better understanding the mechanisms of fermentation inhibition in lignocellulosic hydrolysates. The SH formulated in this work was instrumental for defining the most important inhibitors in the ACH. Major AFEX decomposition products are less inhibitory to yeast fermentation than the products of dilute acid or steam explosion pretreatments; thus, ACH is readily fermentable by yeast without any detoxification.