Separation of Phytochemicals

The goal is to reduce the cost of using cellulase enzymes in the bioethanol process by employing cutting-edge and efficient biochemical technologies. Our current estimate for cellulase ranges from 30 to 50 cents per gallon of ethanol produced. The objective is to reduce cellulase cost to less than 5 cents per gallon of ethanol. This requires a tenfold increase in specific activity or production efficiency or some combination thereof. Nearer-term goals include a threefold increase in cellulase-specific activity (relative to the Trichoderma reesei system) by FY 2005.

Dilute acid pretreatment of hard woods tends to yield residual biomass that is high in native cellulose and very low in hemicellulosic sugars. In the early 1990s, NREL researchers determined that a 90:9:1 mixture of a cellobiohydrolase from T. reesei (CBH I), a thermal-tolerant endoglucanase from A. cellulolyticus (EI), and a ß-D-glucosidase was capable of nearing the performance observed for a leading commercial T. reesei preparation at comparable protein loading (i.e., end-point saccharification of cellulose in pretreated yellow poplar after 120 hours). This discovery was an important breakthrough because the option to engineer cellulase systems for specific pretreated biomass feedstocks became apparent for the first time.

Subcontract:
Develop and Test Improved T. fusca Cellulases
Cornell University (D. Wilson) is evaluating new cellulase mixtures and their application to biomass as well as working to improve T. fusca E3 and E5 by site-directed-mutagenesis.
Protein Engineering

Work at NREL in FY 1997 to 1998, with collaboration from the University of Arkansas (J. Sakon), presented another breakthrough in the cellulase biochemistry field. We showed that the performance (measured as maximum final percent saccharification of biomass) of this ternary system was improved by 13% following site-directed modification of one active site amino acid residue identified from a high resolution X-ray crystallographic structure of EI. We are now pursuing other promising classes of EI mutations — those that modify the chemistry of the biomass interactive surface.

Subcontracts:
Provide High Resolution X-ray Structures
The University of Arkansas (J. Sakon) is working to support site-directed-mutagenesis studies of Acidothermus EI and T. reesei CBH I.

Molecular Modeling of the Interaction of Cellulose with Cellulases and Catalysts
Cornell University (J. Brady) is working to apply molecular mechanics modeling to enhance our understanding of cellulase action. This information is being used to design protein engineering experiments at NREL and elsewhere.

Advanced Cellulase Performance Assays
The analytical membrane-reactor assay for cellulases developed at NREL, the diafiltration saccharification assay (DSA), produces very precise and detailed progress curves for the enzymatic saccharification of cellulosic materials under conditions that mimic those of simultaneous saccharification and fermentation. Recently, extensive enzyme-loading studies have been carried out, using the DSA, on the kinetics of digestion of dilute-acid-pretreated yellow poplar by three different T. reesei cellulase preparations. Analysis of the combined results demonstrates that an approach utilizing "time-to-target" kinetics and plots of the resulting "reciprocal-time-to-target" values versus a function of cellulase loading show highly linear relationships. These linear relationships constitute a convenient "shorthand" method of describing the performance of a given cellulase preparation over a wide range of loading and reaction times.

Subcontract:
Design and Test Improved Assays for Endoglucanases and Exoglucanases
The University of California at Davis (S. Shoemaker) is working to better understand the mechanisms of cellulase action. Methods for evaluating the performance of endoglucanases and exoglucanases acting on soluble and insoluble substrates will be developed.

Trichoderma reesei Proteonomics
For more complex biomass feedstocks, understanding the roles and relationships of component enzymes from the T. reesei cellulase system acting on complex substrates is key to the development of efficient artificial cellulase systems for the conversion of lignocellulosic biomass to sugars. We presume that the following enzymes are important in hydrolyzing biomass: ß-1,4-endoglucanases (EG I, EG II, EG III, and EG V); ß-1,4-cellobiohydrolases (CBH I & CBH II); xylanases (XYN I & XYN II); ß-glucosidase; a-L-arabinofuranosidase; acetyl xylan esterase; ß-mannanase; and a-glucuronidase. At NREL, these enzymes are being fingerprinted from T. reesei broth by 2-D gel electrophoresis and confirmed by direct peptide sequence analysis. The compositions of variably induced cellulase preparations from T.reesei are also being compared by this method and related to the overall activity of these cellulase preparations using the diafiltration saccharification assay.

Recent Publications, Reports, and Presentations
General
Himmel, M.E.; Adney, W.S.; Baker, J.O.; Elander, R.; McMillan, J.D.; Nieves, R.A.; Sheehan, J.; Thomas, S.R.; Vinzant, T.B.; Zhang, M. (1997). "Advanced Bioethanol Production Technologies: A Perspective." Woodward, J.; Saha, B., eds. Fuels and Chemicals from Biomass, ACS Series 666, Washington, DC: American Chemical Society; pp. 2-45.
Himmel, M.E.; Baker, J.O.; Saddler, J., eds. (2001). "Glycosyl Hydrolases for Biomass Conversion." ACS Symposium Series 769, Washington, DC: American Chemical Society: Distributed by Oxford University Press.
Sheehan, J.; Himmel, M.E. (1999). "Enzymes, Energy, and the Environment: Cellulase Development in the Emerging Bioethanol Industry." Biotechnology Progress (15:3); pp. 817-827.

Engineered Cellulase Mixtures
Baker, J.O.; Adney, W.S.; Nieves, R.A.; Thomas, S.R.; Himmel, M.E. (1995). "Synergism in Binary Mixtures of Bacterial and Fungal Cellulases: Endo/Exo, Exo/Exo, and Endo/Endo Interactions." Saddler, J.N.; Penner, M.H. eds. Enzymatic Degradation of Insoluble Polysaccharides, ACS Series 618, Washington, DC: American Chemical Society; pp. 113-141.
Baker, J.O.; Ehrman, C.I.; Adney, W.S.; Thomas, S.R.; Himmel, M.E. (1998) "Hydrolysis of Cellulose Using Ternary Mixtures of Purified Cellulases." Appl. Biochem. Biotechnol. (70:72); pp. 395-403.
Baker, J.O.; King, M.R.; Adney, W.S.; Decker, S.R.; Vinzant, T.B.; Lantz, S.L.; Nieves, R.E.; Thomas, S.R.; Li, L.-C.; Cosgrove, D.J.; Himmel, M.E. (1999). "Investigation of the Cell Wall Loosening Protein Expansion as a Possible Additive in the Enzymatic Saccharification of Lignocellulosic Biomass," Applied Biochemistry and Biotechnology, In Press.
Thomas, S.R.; Laymon, R.A.; Chou, Y.C.; Tucker, M.P.; Vinzant, T.B.; Adney, W.S.; Baker, J.O.; Nieves, R.A.; Mielenz, J.R.; Himmel, M.E. (1995). "Initial Approaches to Artificial Cellulase Systems for Conversion of Biomass to Ethanol." Saddler, J.N.; Penner, M.H., eds. Enzymatic Degradation of Insoluble Polysaccharides, ACS Series 618, Washington, DC: American Chemical Society; pp. 208-236.

Protein Engineering
Godbole, S.; Decker, S.R.; Nieves, R.A.; Adney, W.S.; Vinzant, T.B.; Baker, J.O.; Thomas, S.R.; Himmel, M.E. (1999) "Cloning and Expression of Trichoderma reesei CBH I in Pichia pastoris," Biotechnology Progress (15:3); pp. 828-833.
Himmel, M.E.; Adney, W.S.; Baker, J.O.; Nieves, R.A.; Thomas, S.R. (1996) "Cellulases: Structure, Function, and Application," Wyman, C.E., ed., Handbook on Bioethanol, Washington, DC: Taylor & Francis; pp. 143-161.
Karplus, P.A.; Sakon, J.; Adney, W.S.; Baker, J.O.; Thomas, S.R. (1997) "Polysaccharide Hydrolase Folds: Diversity of Structure and Convergence of Function," Appl. Biochem. Biotechnol. (63:65); pp. 315-326.
Palma, R.; Himmel, M.E.; Liang, G.; Brady, J. (2000) "Molecular Mechanics Studies of Cellulases," Himmel, M.E.; Baker, J.O.; Saddler, J., eds. Glycosyl Hydrolases for Biomass Conversion, ACS Series 769, Washington, DC: American Chemical Society, In press.
Sakon, J.; Adney, W.; Himmel, M; Thomas, S.; Karplus, P. (1996) "Crystal Structure of Thermostable Family 5 Endocellulase EI from Acidothermus cellulolyticus in Complex with Cellotetraose," Biochemistry (35); pp. 10648-10660.
Advanced Cellulase Performance Assays
Baker, J.O.; Vinzant, T.B.; Ehrman, C.I.; Adney, W.S.; Himmel, M.E. (1997) "A Membrane-Reactor Saccharification Assay to Evaluate the Performance of Cellulases Under Simulated SSF Conditions," Appl. Biochem. Biotechnol., (63:65); pp. 585-595.
Vinzant, T.B.; Ehrman, C.I.; Himmel, M.E. (1997) "SSF of Pretreated Hardwoods: Effect of Native Lignin Content." Appl. Biochem. Biotechnol., (62:1); pp. 97-102.