The crystal structure of the catalytic domain of the thermostable endocellulase E1 from Acidothermus cellulolyticus in complex with cellotetraose has been solved by multiple isomorphous replacement and refined at 2.4 A resolution to an R-factor of 0.18 (Rfree = 0.24). E1cd is a member of the 4/7 superfamily of hydrolases, and as expected, its structure is an (alpha/beta)8 barrel, which constitutes a prototype for family 5-subfamily 1 cellulases. The cellotetraose molecule binds in a manner consistent with the expected Michaelis complex for the glycosylation half-reaction and reveals that all eight residues conserved in family 5 enzymes are involved in recognition of the glycosyl group attacked during cleavage. Whereas only three residues are conserved in the whole 4/7 superfamily (the Asn/Glu duo and the Glu from which the name is derived), structural comparisons show that all eight residues conserved in family 5 have functional equivalents in the other 4/7 superfamily members, strengthening the case that mechanistic details are conserved throughout the superfamily. On the basis of the structure, a detailed sequence of physical steps of the cleavage mechanism is proposed. A close approach of two key glutamate residues provides an elegant mechanism for the shift in the pKa of the acid/base for the glycosylation and deglycosylation half-reactions. Finally, purely structural based comparisons are used to show that significant differences exist in structural similarity scores resulting from different methods and suggest that caution should be exercised in interpreting such results in terms of implied evolutional relationships.

Grass species represent a major source of food, feed, and fiber crops and potential feedstocks for biofuel production. Most of the biomass is contributed by cell walls that are distinct in composition from all other flowering plants. Identifying cell wallrelated genes and their functions underpins a fundamental understanding of growth and development in these species. Toward this goal, we are building a knowledge base of the maize (Zea mays) genes involved in cell wall biology, their expression profiles, and the phenotypic consequences of mutation. Over 750 maize genes were annotated and assembled into gene families predicted to function in cell wall biogenesis. Comparative genomics of maize, rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana) sequences reveal differences in gene family structure between grass species and a reference eudicot species. Analysis of transcript profile data for cell wall genes in developing maize ovaries revealed that expression within families differed by up to 100-fold. When transcriptional analyses of developing ovaries before pollination from Arabidopsis, rice, and maize were contrasted, distinct sets of cell wall genes were expressed in grasses. These differences in gene family structure and expression between Arabidopsis and the grasses underscore the requirement for a grass-specific genetic model for functional analyses. A UniformMu population proved to be an important resource in both forward-and reverse-genetics approaches to identify hundreds of mutants in cell wall genes. A forward screen of field-grown lines by near-infrared spectroscopic screen of mature leaves yielded several dozen lines with heritable spectroscopic phenotypes. Pyrolysismolecular beam mass spectrometry confirmed that several nir mutants had altered carbohydrate-lignin compositions.

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Corn stover, the above-ground, non-grain portion of the crop, is a large, currently available source of biomass that potentially could be collected as a biofuels feedstock. Biomass conversion process economics are directly affected by the overall biochemical conversion yield, which is assumed to be proportional to the carbohydrate content of the feedstock materials used in the process. Variability in the feedstock carbohydrate levels affects the maximum theoretical biofuels yield and may influence the optimum pretreatment or saccharification conditions. The aim of this study is to assess the extent to which commercial hybrid corn stover composition varies and begin to partition the variation among genetic, environmental, or annual influences. A rapid compositional analysis method using near-infrared spectroscopy/ partial least squares multivariate modeling (NIR/PLS) was used to evaluate compositional variation among 508 commercial hybrid corn stover samples collected from 47 sites in eight Corn Belt states after the 2001, 2002, and 2003 harvests. The major components of the corn stover, reported as average (standard deviation) % dry weight, whole biomass basis, were glucan 31.9 (2.0), xylan 18.9 (1.3), solubles composite 17.9 (4.1), and lignin (corrected for protein) 13.3 (1.1). We observed wide variability in the major corn stover components. Much of the variation observed in the structural components (on a whole biomass basis) is due to the large variation found in the soluble components. Analysis of variance (ANOVA) showed that the harvest year had the strongest effect on corn stover compositional variation, followed by location and then variety. The NIR/PLS rapid analysis method used here is well suited to testing large numbers of samples, as tested in this study, and will support feedstock improvement and biofuels process research.

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An attempt was made to obtain a high-level production of intact Acidothermus cellulolyticus endoglucanase (E1) in transgenic tobacco plants. The E1 expression was examined under the control of the constitutive and strong Mac promoter or light-inducible tomato Rubisco small sub-unit (RbcS-3C) promoter with its original or Alfalfa Mosaic Virus (AMV) RNA4 5'-untranslated leader (UTL) and targeted to different sub-cellular compartments via transit peptides. The transit peptides included native E1, endoplasmic reticulum, vacuole, apoplast, and chloroplast. E1 expression and its stability in transgenic plants were determined via E1 activity, protein immunoblotting, and RNA gel-blotting analyses. Effects of sub-cellular compartments on E1 production and its stability were determined in transgenic tobacco plants carrying one of six transgene expression vectors, where the E1 was under the control of Mac promoter, mannopine synthase transcription terminator, and one of the five transit peptides. Transgenic tobacco plants with an apoplastic transit peptide had the highest average E1 activity and protein accumulation, which was about 0.25% of total leaf soluble proteins estimated via E1 specific activity and protein gel blots. Intercellular fluid analyses confirmed that E1 signal peptide functioned properly in tobacco cells to secret E1 protein into the apoplast. By replacing RbcS-3C UTL with AMV RNA4 UTL E1 production was enhanced more than twofold, while it was less effective than the mannopine synthase UTL. It was observed that RbcS-3C promoter was more favorable for E1 expression in transgenic plants than the Mac promoter. E1 activity in dried tobacco seeds stored one year at room temperature was 45% higher than that observed immediately after harvesting, suggesting that E1 protein can be stored at room temperature for a long period. E1 stability in different sub-cellular compartments and the optimal combination of promoter, 5'-UTL, and sub-cellular compartmentation for heterologous protein production in transgenic plants are discussed.

New, rapid, and inexpensive methods that monitor the chemical composition of corn stover and corn stover-derived samples are a key element to enabling the commercialization of processes that convert stover to fuels and chemicals. These new techniques combine near infrared (NIR) spectroscopy and projection to latent structures (PLS) multivariate analysis to allow the compositional analysis of hundreds of samples in 1 d at a cost of about $10 each. The new NIR/PLS rapid analysis methods can also be used to support a variety of research projects that would have been too costly to pursue by traditional methods.

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