Biomass Depolymerization programs
Enhanced Conversion of Lignocellulose to Biofuels: Bioprocess Optimization from Cellulose Hydrolysis to Product Fermentation
This program is developing new experimental systems to study cellulosome degradation of cellulosic biomass. This includes discovering new thermophilic organisms as enzyme sources and/or for biofuel production, protein engineering and kinetic modeling of improved cellulases, cellular engineering for improved solvent tolerance, and bioprocess engineering to optimize fermentation. The team seeks to understand the regulation of cellulosome composition, expression and assembly; probe activities of various cellulosomal components and assemblies; and identify dynamics of lignocellulosic degradation by using single molecule and atomic force microscopy.
Our program addresses the major bottlenecks in the production of biofuels from lignocellulosic feedstocks. The program has several inter-related components that interface closely with complementary research performed throughout the EBI. The program includes improving cellulolytic activity of cellulase enzymes by protein engineering, developing a combined biological/chemical approach to convert the products of the ABE fermentation to biofuels, and integrating cellular and bioprocess engineering to optimize biofuels production by fermentation.
Our program addresses the major bottlenecks in the production of biofuels from lignocellulosic feedstocks. The program has several inter-related components that interface closely with complementary research performed throughout the EBI. The program includes improving cellulolytic activity of cellulase enzymes by protein engineering, developing a combined biological/chemical approach to convert the products of the ABE fermentation to biofuels, and integrating cellular and bioprocess engineering to optimize biofuels production by fermentation. Our accomplishments in 2013 include successfully creating fungal cellulases expressed in S. cerevisiae that exhibit properties comparable to their counterparts expressed in N. crassa. We also studied cellobiose inhibition of CBHI, and our results provide the first direct evidence of how cellobiose hinders catalysis. We also engineered C. acetobutylicum strains to produce isopropanol and hexanol for catalytic upgrading to higher-chain diesel compounds and lubricants. This work demonstrates the tunability of this platform organism for a variety of potential end-products. We will build upon these and other achievements and contribute to the future of sustainable fuels and chemicals.
Our program addresses the major bottlenecks in the production of biofuels from lignocellulosic feedstocks. Key activities include protein engineering to improve the cellulolytic activity of cellulase enzymes, modeling of cellulase kinetics, development of a combined biological/chemical process to convert the products of the acetone-butanol-ethanol (ABE) fermentation to biofuels, cellular engineering for improved solvent tolerance, and bioprocess engineering to optimize biofuels production by fermentation.
Notable accomplishments in 2012 include developing a detailed mechanistic model of cellobiohydrolase action and use of the model to elucidate the rate limitations imposed upon cellobiohydrolase enzymes as they catalyze the hydrolysis of crystalline cellulose alone and in concert with endoglucanases. This mechanistic model was combined with a multi-substrate model of microbial growth to evaluate the economic impact of cellobiose transporters and cellobiose utilization on ethanol production for different processing configurations (e.g., simultaneous saccharification and fermentation [SSF] versus separate hydrolysis and fermentation [SHF]). We also made notable progress in cellulase engineering, developing a broadly applicable cellulase mutagenesis platform and applying it to increase the operating temperature and thermostability of the fungal glycosyl hydrolase Cel7A.
In the area of lignocellulose pretreatment, we developed a model for determining the optimal conditions (e.g., time, temperature, biomass loading) for ionic-liquid pretreatment of Miscanthus x giganteus to maximize the effectiveness of subsequent depolymerization by cellulases. Finally, in collaboration with Dean Toste and co-workers at EBI, we demonstrated a new approach that integrates biological and chemocatalytic routes to efficiently convert ABE fermentation products into fungible hydrocarbon fuels at high yields from biomass. This process provides a route to selectively produce gasoline, jet, and diesel fuels from lignocellulosic and cane sugars at yields near their theoretical maxima.
The Clark and Branch group isolated cellulases from thermophilic and other microbes, then engineered them to be more active and stable at high temperatures. To aid in rational design of enzymes, they developed models of carbohydrate binding to cellulase. Biofuels can be toxic to the microbes that produce them, so Clark’s group also has been exploring ways to increase microbial tolerance. They showed that microbes could grow in higher concentrations of ethanol if a gene were introduced that helps to stabilize proteins.
The program now includes new approaches to pretreat biomass using ionic liquids, the discovery of extremophiles as sources of glycosidic enzymes, and protein engineering and kinetic modeling of cellulose activity. The Clark and Branch lab also has developed a detailed mechanistic model of cellulose hydrolysis and has discovered a novel hyperthermophilic cellulase from a cellulolytic archaeal consortium. The group has generated multiple libraries of cellulases by protein engineering and are screening those libraries for improved properties, such as greater thermostability.
The Clark and Blanch lab, in collaboration with the Frank Robb lab at the University of Maryland, cultivated communities of organisms previously collected from hot springs in eastern Russia and the continental U.S. to find novel thermophiles that either produce biofuels (ethanol and/or butanol) or have thermostable enzymes that degrade cellulose. This approach has produced several cultures that degrade cellulose at high temperatures. Isolating and characterizing those enzymatic units is progressing.
Published in 2014
Engineering Clostridium Acetobutylicum for Production of Kerosene and Diesel Blendstock Precursors, S. Bormann, Z. C. Baer, S. Sreekumar, J. M. Kuchenreuther, F. D. Toste, H. W. Blanch, D. S. Clark, Metabolic Engineering, V. 55, pp. 124-130, July 18, 2014.
Chemocatalytic Upgrading of Tailored Fermentation Products Toward Biodiesel, S. Sreekumar, Z. C. Baer, E. Gross, S Padmanaban, K. Goulas, G. Gunbas, S. Alayoglu, H. Blanch, D. S. Clark, and F. D. Toste, ChemSusChem, V. 7 (9), pp. 2445–2448, July 15, 2014.
The Importance of Pyroglutamate in Cellulase Cel7A, C. M. Dana, A. Dotson-Fagerstrom, C. M. Roche, S. M. Kal, H. A. Chokhawala, H. W. Blanch, D. S. Clark, Biotechnology Bioengineering, 111, pp. 842–847, January 28, 2014.
Poly (styrene-b-dimethylsiloxane-b-styrene) Membranes in Pervaporation for In Situ Product Recovery During Fermentation, C. Shin, Z. C. Baer, A. E. Ozcam, D. S. Clark, N. P. Balsara, Bulletin of the American Physical Society, 51(9), 2014.
Published in 2013
A Single-Molecule Analysis Reveals Morphological Targets for Cellulase Synergy, J. M. Fox, P. Jess, R. B. Jambusaria, G. M. Moo, J. Liphardt, D. S. Clark, H. W. Blanch, Nature Chemical Biology 9, pp. 356-361, doi: 10.1038/nchembio,1227, April 7, 2013.
The Importance of Pyroglutamate in Cellulase Cel7a, Craig M. Dana, Alexandra Dotson-Fagerstrom, Christine M. Roche, Sarala M. Kal, Harshal A. Chokhawala, Harvey W. Blanch, Douglas S. Clark, Biotechnology and Bioengineering, doi: 10.1002/bit.25178, December 30, 2013
Published in 2012
Delignification of Miscanthus by Extraction, S. Padmanabhan, E. Zaia, K. Wu, H. W. Blanch, D. S. Clark, A. T. Bell, and J. M. Prausnitz, Separation Science and Technology 47. 370.
Initial- and Processive-Cut Products Reveal Cellobiohydrolase Rate Limitations and the Role of Companion Enzymes, J. M. Fox, S. E. Levine, D. S. Clark, and H. W. Blanch, Biochemistry 51, 442.
Extraction of Lignins from Aqueous-Ionic Liquid Mixtures by Organic Solvents, Q. Xin, K. Pfeiffer, J. M. Prausnitz, D. S. Clark, and H. W. Blanch, Biotechnology & Bioengineering 109, 346.
Escherichia coli for Biofuel Production: Bridging the Gap from Promise to Practice, S. Huffer, C. M. Roche, H. W. Blanch, and D. S. Clark, Trends in Biotechnology 30, 538.
A Model for Optimizing the Enzymatic Hydrolysis of Ionic Liquid-Pretreated Lignocellulose, K. Shill, K. Miller, D. S. Clark, and H. W. Blanch, Bioresource Technology, 126, 290 (2012).
Biased clique shuffling reveals stabilizing mutations in cellulase Cel7A, Craig M. Dana, Poonam Saija, Sarala M. Kal, Mara B. Bryan, Harvey W. Blanch, Douglas S. Clark, Biotechnology & Bioengineering, http://dx.doi.org/10.1002/bit.24708, Sept. 14, 2012.
An Evaluation of Cellulose Saccharification and Fermentation with an Engineered Saccharomices cerevisiae Capable of Cellulose and Zylose Utilization, Jerome Fox, Seth Levine, Harvey Blanch, Douglas Clark, Biotechnology Journal, doi: 10.1002/biot.201100209, March 2, 2012.
Integration of Chemical Catalysis with Extractive Fermentation to Produce Fuels, Pazhamalai Anbarasan, Zachary C. Baer, Sanil Sreekumar, Elad Gross, Joseph B. Binder, Harvey W. Blanch, Douglas S. Clark, and F. Dean Toste, Nature, doi: 10.1038/nature11594, November 8, 2012.
Published in 2011
An Evaluation of Cellulose Saccharification and Fermentation with an Engineered Saccharomyces cerevisiae Capable of Cellobiose and Xylose Utilization, Jerome Fox, Seth Levine, Harvey Blanch, Douglas Clark, Biotechnology Journal, doi:10.1002/biot.201100209, December 29, 2011.
Initial- and Processive-cut Products from Cellobiohydrolase-catalyzed Hydrolysis of Cellulose Reveal Rate-limiting Steps and Role of Companion Enzymes, Jerome Fox, Seth Levine, Douglas Clark, Harvey Blanch, Biochemistry, doi: 10.1021/bi2011543, November 21, 2011.
A Mechanistic Model for Rational Design of Optimal Cellulase Mixtures, Seth Levine, Jerome Fox, Douglas Clark, Harvey Blanch, Biotechnology and Bioengineering, 108(11), pp. 2561-2570, doi: 10.1002/bit.23249, November 2011.
Extraction of Lignins from Aqueous-Ionic Liquid Mixtures by Organic Solvents, Qin Xin, Katie Pfeiffer, John Prausnitz, Doug Clark, Harvey Blanch, Biotechnology and Bioengineering, doi:10.1002/bit.24337, October 19, 2011.
Green Fluorescent Protein as a Screen for Enzymatic Activity in Ionic Liquid-aqueous Systems for in situ Hydrolysis of Lignocellulose, Paul Wolski, Douglas Clark, Harvey Blanch, Green Chemistry, doi:10.1039/C1GC15691H, October 5, 2011.
High-Throughput In Vitro Glycoside Hydrolase (HIGH) Screening for Enzyme Discovery, Dae-Wan Kim, Harshal Chokhawala, Matthias Hess, Craig Dana, Zachary Baer, Alexander Sczyrba, Edward Rubin, Harvey Blanch, Douglas Clark, Angewandte Chemie, doi: 10.1002/anie.201104685, September 16, 2011.
The Role of Alcohols in Growth, Lipid Composition, and Membrane Fluidity of Yeast, Sarah Huffer, Melinda Clark, Jonathan Ning, Harvey Blanch, Douglas Clark, Applied Environmental Microbiology, doi:10.1128/AEM.00694-11, July 22, 2011.
Identification and Characterization of a Multidomain Hyperthermophilic Cellulase from an Archaeal Enrichment, Joel Graham, Melinda Clark, Dana Nadler, Sarah Huffer, Harshal Chokhawala, Sara Rowland, Harvey Blanch, Douglas Clark, Frank Robb, Nature Communications, doi:10.1038/ncomms1373, July 5, 2011.
Ionic Liquid Pretreatment of Cellulosic Biomass: Enzymatic Hydrolysis and Ionic Liquid Recycle, Kierston Shill, Sasisanker Padmanabhan, Qin Xin, John Prausnitz, Douglas Clark, Harvey Blanch, Biotechnology and Bioengineering, 108(3), pp. 511-520, doi: 10.1002/bit.23014, March 2011.
Published in 2010
Binding Modules Alter the Activity of Chimeric Cellulases: Effects of Biomass Pretreatment and Enzyme Source , Tae-Wan Kim, Harshal Chokhawala, Dana Nadler, Harvey Blanch, Douglas Clark, Biotechnology and Bioengineering, June 21, 2010.
A Mechanistic Model of the Enzymatic Hydrolysis of Cellulose, Seth Levine, Jerome Fox, Harvey Blanch, Douglas Clark, Biotechnology and Bioengineering, May 18, 2010.
Elucidating Mechanisms of Solvent Toxicity in Ethanologenic Escherichia coli, Cong Trinh, Sarah Huffer, Melinda Clark, Harvey Blanch, Douglas Clark, Biotechnology and Bioengineering, 106(5): pp. 721-730, March 26, 2010.
Published in 2009
Tying Up the Loose Ends: Circular Permutation Decreases the Proteolytic Susceptibility of Recombinant Proteins, Timothy Whitehead, Lisa Bergeron, Douglas Clark, Protein Engineering Design and Selection, 22(10): pp. 607-613, June 8, 2009.