In plants, the first step in myo-inositol synthesis is the cyclization of d-G1c-6-P to myo — inositol-1-P (Ino-1P) by lL-myo-inositol 1-phosphate synthase. In Arabidopsis, two func­tional isoforms were reported, At4g39800 and At2g22240 (454). The second step involves dephosphorylation of Inol-P to myo-inositol by myo-inositol monophosphatase (IMPase; EC 3.1.3.25) (455). Distinct multiple but highly conserved IMPase isoforms are found in each plant species. Three IMPases were identified in tomato (456). In Arabidopsis, a con­served IMPase-like-protein (At3g02870) was proposed by Glilaspi to act as IMPase; however, biochemical and genetic data indicate that At3g02870 encodes L-Gal-1-P phosphatase (457, 458). It is possible that other IMPase-like proteins in Arabidopsis (for example, At1g31190, At4g39120) encode the Ino-1P phosphatase activity to form myo-inositol. The identification of the true IMPase gene product is critical to evaluate what controls the pathway to shunt Ino-1P to the myo-inositol oxidation pathway. Free myo-inositol is oxidized by inositol oxy­genase (MIOX; E. C. 1.13.99.1) to D-GlcA. Arabidopsis contains four Miox isoforms (453).

It would be interesting to determine if the myo-inositol oxidation pathway operates inde­pendently of the pathway leading to synthesis of UDP-GlcA from UDP-Glc. This knowledge could aid in determining which flux of sugars the plant uses to facilitate wall synthesis in specific tissues. For example, myo-inositol in seed is stored as phytic acid (inositol hexaphos — phate). During germination, phosphatases provide a rapid source of inositol which is con­verted, in part, to GlcA. Hence, this would provide a source of UDP-GlcA for wall pectin synthesis. However, during germination, rapid synthesis of L-ascorbate from myo-inositol also occurs. The relationship and coordination of the supply of sugars to wall glycans and to ascorbate synthesis must be better understood at all stages of growth.

These analyses have been most useful in predicting the outcome of various manipulations in the lignin-forming pathway. That is, previous studies, whereby monolignol 1 and 3 formation could be induced in loblolly pine (Pinus taeda) cell suspension cultures, enabled us to gain important insights into factors controlling metabolic flux to both p-coumaryl (1) and coniferyl (3) alcohols (34, 35). Thus, by increasing levels of available sucrose, the monolignol-forming pathway could be induced, with the cells secreting the monolignols (in the presence of an H2O2-scavenger) into the culture medium.

The data (based on both measuring various pathway metabolite levels and transcript pro­files) provided quite informative insights: the first was that (regulation of) carbon allocation to the pathway was controlled upstream through the amounts of Phe (6) being made avail­able, as well as through the differential activities of both cinnamate-4-hydroxylase (C4H) and p-coumarate-3-hydroxylase (pC3H). Furthermore, metabolite analyses also indicated that formation of both p-coumaryl (1) and coniferyl (3) alcohols could be differentially in­duced, suggesting the existence of distinct metabolic control over segments (i. e., H versus G) within the monolignol/lignin forming processes through differential modulation of pC3H activity. Beyond the hydroxylation steps, other downstream enzymatic steps (see Figure 7.1) were not considered to be rate limiting, at least under the conditions employed in the studies. [Of course, any enzymatic step becomes rate limiting if abolished or “knocked out.”] Tran­scriptional profiling data of each of the known steps involved in monolignol biosynthesis (available at that time) also appeared to support this analysis and interpretation (35).

One of the most exciting discoveries in recent years, as regards cell wall formation, is that of the roles of two transcription factors [SND1 (also called NST3) and NST1]. These are responsible for (secondary wall formation) in fibers of Arabidopsis (198, 199), and are specifically expressed in interfascicular fibers and xylary fibers as shown using the GUS reporter gene strategy (198,199). Analysis of the SND1 (NST3) knockout line, however, did not initially indicate any anatomical differences when compared to the wild type, this being attributed to possible genetic redundancy. To overcome this problem, a dominant repression strategy (198) was next used where SND1 (NST3) was fused to the EAR repression domain (283). Fifteen of the 64 transgenic plants obtained displayed a phenotype that was unable to stand upright. Cross-sections of the stems showed that the interfascicular fibers and xylary fibers were very thin, due to a lack of secondary wall development; the cell walls of the vessels by comparison were unaffected.

Analysis of SND1 (NST3) and NST1 promoter activity also indicated that both transcrip­tion features were expressed in fibers suggesting that both may be involved in secondary wall thickening (199). A double mutant, nst1-1 nst3-1, was next generated, with the result­ing phenotypes such that the plants obtained were unable to remain upright after reaching ~15 cm in length (Figure 7.13F). Stem cross-sections also indicated that the interfascicular fibers were not autofluorescent (an indication of lignification) compared to wild type, and ultrastructural analyses established the absence of secondary cell walls in the fibers; again vessels were unaffected as regards normal secondarycell wall development. Quantitative real time PCR analyses also indicated that various genes involved in secondary wall biosynthesis were suppressed (198,199), these included those involved in monolignol (CCOMT, At4CL1, AtOMTl, AtCCR1), as well as cellulose (irx3, irx5) and xylan (fra8) biosynthesis. This is a very important discovery, not only that fiber secondary wall formation is under control of transcriptional factors, but also that the plant lines are again unable to compensate in any effective manner for the defects introduced.

There are two major approaches to hydrolysis of cellulose, acid hydrolysis, and enzymatic hydrolysis. The enzymatic process is poorly understood and must contain the solution to the recalcitrant nature of cellulosic degradation. The enzymes can be modeled, as well as their interactions with cellulose and even the process of enzymatic hydrolysis. The techniques that will probe the processes and mechanisms are numerous and range from reduced models to all-atom QM/MM and thermodynamic integration. Using reduced models, the structural stabilities and solvation free energies can be determined quickly. Normal mode and elastic network models and quasiharmonic analysis can probe the major structural modes of motion of cellulose, cellulases, xylans, lignins, and their mutual complexes. Mutational studies, using thermodynamic integration, can be performed to reveal the effects on structure and on kinetic behaviors, and even on reaction energetics and mechanisms. Umbrella sampling is a key player in understanding the binding affinities of different binding or catalytic domains on cellulose, or the relative binding affinities on different faces of cellulose or even on different locations of the same face. QM/MM is a tool for probing the hydrolysis reaction inside a cellulase catalytic site. This method is at the stage of development that performance is sufficient and the QM approximations are good enough to follow a reaction quantum mechanically while treating the non-reactive portion of the system classically and have reasonable answers for not much higher computational cost than pure classical simulations. It is expected that exceptionally useful information about the release of energy from reaction, and the accompanying structural changes will come from these numerical experiments.

The steered molecular dynamics, targeted MD, and pulling methods are the tools of choice for initial examination of the process of decrystallization of the cellulose fibers into cellodex­trin chains suitable for hydrolysis to mono — and disaccharides. These kinds of numerical experiments can suggest the energy barriers associated with decrystallization, and suggest more detailed studies such as obtaining PMF profiles from umbrella sampling runs, or free energies of decrystallization from Jarzynski pulling experiments. Beyond that, details about how the solvent plays a role in all the aforementioned processes can be carefully quantified and help to select the most likely and deselect unlikely mechanisms.

The most studied aerobic cellulolytic microorganism is the fungus, Hypocrea jecorina, orig­inally called Trichoderma reesei. It was isolated and studied by Drs Reese and Mandels at the

Army Quartermaster Lab in Natick, MA, during World War II, because it was degrading the cotton fabrics used by the army for tents, gun straps, etc. on islands in the Pacific Ocean (56). The original goal of this work was to find cellulase inhibitors, which was not achieved, as only the toxic ions, Hg and Ag, are good inhibitors. However, this group also carried out many studies on the organism and its crude cellulase, which then led to the development of high producing mutant strains by Dr Eveleigh that were used to develop the strains used for industrial cellulase production by several companies (57). The most abundant cellulase produced by T. reesei is the reducing end-specific exocellulase, Cel7A (cellobiohydrolase I), which makes up about 70% of the cellulase protein secreted by T. reesei (58). The next most abundant cellulase is Cel6A (CBH II), which makes up a further 10% of T. reesei secreted cellulase. T. reesei crude cellulase contains seven endoglucanases of which Cel7B (EGLI) is the most abundant. In addition, Cel5A (EGLII), Cel12A (EGLIII), Cel61A (EGLIV), Cel45A (EGLV), Cel5B, and Cel61B are also present in T. reesei secreted cellulase. Most of the T. reesei cellulases contain a family I CBM except Cel5B, Cel12A, and Cel61B. It is not clear why T. reesei produces so many endocellulases, but Cel12A was shown to have expansin activity as well as cellulase activity (59). Expansin is present in plants and it appears to disrupt the hydrogen bonds that bind different carbohydrate chains together in plant cell walls, so that it may make the chains more accessible to hydrolytic enzymes. The least studied endocellulase is Cel61A, which has extremely low cellulase activity. It is quite surprising that when a set of thermophilic fungal cellulases were screened for the ability to stimulate the activity of T. reesei crude cellulase, a number of them were able to increase it about threefold and the component that was most active in giving this stimulation was a family 61 enzyme (60). Little is known about the role of Cel45A in cellulose degradation (61). Another protein se­creted by T. reesei is swollenin, which is a low molecular weight protein that has no catalytic activity but appears to disrupt the structure of cellulose microfibers, possibly by breaking hydrogen bonds (62).

Most of the T. reesei cellulases are glycosylated and glycosylation appears to protect the cellulases from proteolysis (63). The linker peptide is particularly susceptible to proteolysis and T. reesei secretes proteases (64), so that protection from proteolysis maybe an important role for the O-linked glycosylation found on the linker peptide (65). The role of the N-linked glycosylation on the CD is unclear at this time. There is a great deal of heterogeneity in the glycosylation of any given cellulase and this causes heterogeneity of each enzyme during gel electrophoresis and column chromatography (66).

There have been extensive studies of the regulation of cellulase synthesis in T. reesei and it appears that regulation is complex (67). Glucose strongly represses cellulase synthesis and the (3-1,2-linked glucose disaccharide, sepharose, induces synthesis. A number of transcription factors have been identified in T. reesei, which can bind to cellulase promoters, and some of these are activators and some are repressors. The exact mechanisms that regulate cellulase synthesis are still not completely understood.

Another category of solvent pretreatment involves the use of cellulose-dissolving solvents, such as cadoxen, concentrated mineral acids, DMSO, and zinc chloride (10, 12). While these agents can be effective at directly releasing sugars from the carbohydrate fractions of biomass and/or producing a solid residue containing cellulose that is highly digestible by enzymes, the use of such solvents in pretreatment processes for the production of fuels and commodity chemicals from biomass will be challenging due to the expense of such catalysts, catalyst recycle requirements, and the requirement for clean process streams for subsequent biological conversions.

14.5.4 Supercritical fluid pretreatments

Biomass pretreatment processes using supercritical fluids to extract lignin from biomass feedstock have been investigated. A number of different supercritical fluids (alone or in mixtures) have been investigated, although the most common approaches utilize water, carbon dioxide, or ammonia (14, 68). While supercritical pretreatment conditions can effectively remove lignin and produce pretreated biomass that exhibits good enzymatic digestibility, the economic viability and practical operation of processes at supercritical operating conditions have not been effectively demonstrated. Of greatest concern are the extremely high-pressure requirements (generally above 10 MPa) of these processes.

In the aqueous gel matrix of the primary wall polysaccharide chains interact non­covalently to form a continuous three-dimensional network. The polysaccharide chains in the gel have two types of domains: open, hydrated, unassociated regions and regions where the complementary conformations of two or more chains permit association over restricted segments (junction zones) (78). The forces stabilizing these junction zones are intermolecular hydrogen bonds or ionic forces. The capacity of matrix polysaccharides to form junction zones is variable and depends on the stereoregularity of the chain, determined by the monosaccharide and linkage sequence, the presence of bulky side chains and the proximity of mutually repulsive charged residues. The physical characteristics of the gel matrix will depend on the lengths and numbers of junction zones. The non-cellulosic wall polysaccharides generally have features that make them potential gel-forming polymers. They have linear backbones, are more or less soluble in water, and in contrast to the cellulosic fibrillar phase, show conformational irregularities. The non-covalent interactions of the non-cellulosic polysaccharides with one another and the surfaces of cellulosic microfibrils are important in determining the cohesivity of cell walls as is discussed in Section 4.4 — Molecular architecture of plant cell walls.

In addition to the functional strengthening of walls by non-covalent interactions be­tween individual matrix polymers and matrix polymers and the cellulosic microfibrillar phase, these interactions are reinforced by direct covalent associations between polysac­charides, polysaccharides and lignin, polysaccharides and protein, and between proteins, as well as indirect associations between polysaccharides and polysaccharides and lignin through covalently-linked bridging molecules. The chemistry of this cross-linking is described in the following sections.

Recently, work from the Ulvskov and Geshi groups (274) has provided strong evi­dence that these investigators have identified two Arabidopsis thaliana RG-II-a-D-1,3- xylosyltransferases (RG-II-a1,3XylTs) (274). Following the identification of a novel family of 27 putative Arabidopsis thaliana glycosyltransferases (215) and through a series ofbioin- formatic analyses aimed at identifying novel plant cell wall biosynthetic glycosyltransferases with a predicted Type II membrane topology (359), two of these genes, named RGXT1 (At4g01770) and RGXT2 (At4g01750) were shown to encode proteins with characteris­tics consistent with a function as RG-II-a1,3XylTs. RGXT1 and RGXT2 encode proteins of 361 and 367 amino acids, respectively, share 90% sequence identity, and are members of GT-family 77 (138) (http://afmb. cnrs-mrs. fr/CAZY/). Two additional Arabidopsis genes, At4g01220 and At1g56550, are 68-75% identical to RGXT1 and RGXT2 (274). Expression of truncated soluble forms ofRGXT1 andRGXT2 inbaculovirus-transfected insect cells and enzyme assays using diverse radiolabeled nucleotide-sugars and free monosaccharide accep­tors demonstrated that the expressed proteins catalyze the transfer ofXyl from UDP-a-D-Xyl onto fucose. Biochemical analyses of the synthesized product using specific xylosidases and NMR spectroscopy indicated that the xylose was transferred onto the fucose in an a-1,3- linkage. Based on these results the authors hypothesized that RGXT1 and RGXT2 function in the synthesis of the RG-II side chain A that contains 2- O-methyl-D-Xyl attached in an a1,3-linkage to a-L-Fuc. Acceptor specificity studies demonstrated that both enzymes pre­ferred l-Fuc with an a-anomeric linkage and disaccharide acceptors with Fuc attached at the position 4, rather than at the 2 or 3 position, to another glycosyl residue; all characteris­tics consistent with the structure of RG-II (159). Importantly, RG-II isolated from RGXT1 and RGXT2 mutant walls, but not RG-II from wild type Arabidopsis walls, served as an acceptor for the enzyme, providing strong evidence that RGXT1 and RGXT2 function in RG-II synthesis (274) and providing strong support for the function of RGXT1 and RGXT2 as RG-II-a1,3XylTs. The lack of a clear difference in the structure of RG-II isolated from walls of RGXT1 and RGXT2 mutants compared to wild type walls, however, is perplexing and leaves open the question of whether there is gene redundancy, thus requiring a double (or more) gene knockout mutant to see a phenotype. Alternatively, the question remains as to whether RGXT1 and RGXT2 may have additional or alternative functions in the synthesis of some other, yet to be identified, wall polysaccharide structure. Further studies of RGXT1 and RGXT2 and related genes should clarify their role(s) in pectin synthesis.

GDP-$-L-gluclose gulose (GDP-Gul)

GDP-Gal is a major precursor for the synthesis of ascorbic acid in plants, and relatively low amounts of L-Gal are found in plant glycans. A GDP-Man 3′,57 epimerase activity first identified in the Neufeld’s laboratory (493) epimerizes GDP-Man into GDP-Gal. Careful biochemical analyses of the specific activity of recombinant Arabidopsis At5g28840 (494) and rice (497) protein demonstrate that the enzyme can convert GDP-Man to both GDP-Gul and GDP-Gal (496). A crystal structure of the enzyme was recently obtained (497).

For a period now approximating well over 30 years, NMR spectroscopy has been applied to the study of various lignin isolates (71, 131, 132, 154-177). In this way, it has been possible, together with the study of various dimeric monolignol-derived products, to identify a number of the most abundant substructures in lignins. Many of these were previously shown in Figure 7.2D, for coupling of the H-, G-, and S-derived monolignols, respectively, with the so-called 8-O-4′ interunit linkages generally being acknowledged as the most prevalent. There are, however, still major limitations in current NMR spectroscopic analyses. One is in the inability to determine the sequences ofinterunit linkages within thebiopolymers, because it is not yet currently possible (using natural abundance 13C) to readily go beyond the ether interunit linkages (e. g., 8- O-4′,4- O-5′ linkages) to adjacent flanking substructure(s). A second major difficulty is that of the polymeric nature of the lignins: in general, the spectral band width lines for polymers can be very broad, due to molecular weight (size), polydispersity of samples, molecular aggregation and molecular heterogeneity, etc. For high molecular weight entities, such as with lignins, the molecules can thus experience slow tumbling which, in turn, results in very large transverse magnetization rates (efficient T2 relaxation) leading to broad spectral band width lines. Furthermore, given that all lignin isolation procedures result in preparations that are polydisperse, this — together with possible molecular heterogeneity (= different substructures) in the polymeric chains — can lead to

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very small changes in chemical shifts at the local bonding sites and hence signal broadening. A further complication is the ability of the lignins to aggregate/self-associate which further confounds the spectroscopic analyses.

On the other hand, it is also well known that the more mobile functionalities in polymeric backbones can readily be detected [e. g., acetate groups ofxanthan gum preparations (178)], whereas the polymeric backbones are more extensively line-broadened. A similar situation also presumably holds for different segments of the lignin polymer chains. Furthermore, even with observable resonances by various forms of NMR analyses, quantification of the signals relative to the entire polymeric entities can be quite problematic, again further il­lustrating issues as regards precise quantification. As a further caveat, chemical degradation protocols can be used to identify, detect, and quantify various lignin substructures which cannot readily be detected by NMR spectroscopic techniques. Thus, NMR spectroscopy, while a most powerful technique, currently only provides a very incomplete assessment of the nature of the lignin macromolecule(s). For these reasons, the question of lignin struc­tural analysis has thus been limited to date in attempts to identify both interunit linkages, and less precisely to estimations of probable amounts of the distinct substructures, in the various lignin-enriched isolates — at least, for those that can be distinguished/identified/ detected.