Five amino acid mismatches in the zinc-finger domains of Cellulose Synthase 5 and Cellulose Synthase 6 cooperatively modulate their functional properties by controlling homodimerization in Arabidopsis.

Cellulose synthase 5 (CESA5) and CESA6 are known to share substantial functional overlap. In the zinc-finger domain (ZN) of CESA5, there are five amino acid (AA) mismatches when compared to CESA6. These mismatches in CESA5 were replaced with their CESA6 counterparts one by one until all were replaced, generating nine engineered CESA5s. Each N-terminal enhanced yellow fluorescent protein-tagged engineered CESA5 was introduced to prc1-1, a cesa6 null mutant, and resulting mutants were subjected to phenotypic analyses. We found that five single AA-replaced CESA5 proteins partially rescue the prc1-1 mutant phenotypes to different extents. Multi-AA replaced CESA5s further rescued the mutant phenotypes in an additive manner, culminating in full recovery by CESA5G43R + S49T+S54P+S80A+Y88F. Investigations in cellulose content, cellulose synthase complex (CSC) motility, and cellulose microfibril organization in the same mutants support the results of the phenotypic analyses. Bimolecular fluorescence complementation assays demonstrated that the level of homodimerization in every engineered CESA5 is substantially higher than CESA5. The mean fluorescence intensity of CSCs carrying each engineered CESA5 fluctuates with the degree to which the prc1-1 mutant phenotypes are rescued by introducing a corresponding engineered CESA5. Taken together, these five AA mismatches in the ZNs of CESA5 and CESA6 cooperatively modulate the functional properties of these CESAs by controlling their homodimerization capacity, which in turn imposes proportional changes on the incorporation of these CESAs into CSCs.

Xylan Is Critical for Proper Bundling and Alignment of Cellulose Microfibrils in Plant Secondary Cell Walls.

Plant biomass represents an abundant and increasingly important natural resource and it mainly consists of a number of cell types that have undergone extensive secondary cell wall (SCW) formation. These cell types are abundant in the stems of Arabidopsis, a well-studied model system for hardwood, the wood of eudicot plants. The main constituents of hardwood include cellulose, lignin, and xylan, the latter in the form of glucuronoxylan (GX). The binding of GX to cellulose in the eudicot SCW represents one of the best-understood molecular interactions within plant cell walls. The evenly spaced acetylation and 4-O-methyl glucuronic acid (MeGlcA) substitutions of the xylan polymer backbone facilitates binding in a linear two-fold screw conformation to the hydrophilic side of cellulose and signifies a high level of molecular specificity. However, the wider implications of GX-cellulose interactions for cellulose network formation and SCW architecture have remained less explored. In this study, we seek to expand our knowledge on this by characterizing the cellulose microfibril organization in three well-characterized GX mutants. The selected mutants display a range of GX deficiency from mild to severe, with findings indicating even the weakest mutant having significant perturbations of the cellulose network, as visualized by both scanning electron microscopy (SEM) and atomic force microscopy (AFM). We show by image analysis that microfibril width is increased by as much as three times in the severe mutants compared to the wild type and that the degree of directional dispersion of the fibrils is approximately doubled in all the three mutants. Further, we find that these changes correlate with both altered nanomechanical properties of the SCW, as observed by AFM, and with increases in enzymatic hydrolysis. Results from this study indicate the critical role that normal GX composition has on cellulose bundle formation and cellulose organization as a whole within the SCWs.

The N-terminal zinc finger of CELLULOSE SYNTHASE6 is critical in defining its functional properties by determining the level of homodimerization in Arabidopsis.

Primary cell wall cellulose is synthesized by the cellulose synthase complex (CSC) containing CELLULOSE SYNTHASE1 (CESA1), CESA3 and one of four CESA6-like proteins in Arabidopsis. It has been proposed that the CESA6-like proteins occupy the same position in the CSC, but their underlying selection mechanism remains unclear. We produced a chimeric CESA5 by replacing its N-terminal zinc finger with its CESA6 counterpart to investigate the consequences for its homodimerization, a crucial step in forming higher-order structures during assembly of the CSC. We found that the mutant phenotypes of prc1-1, a cesa6 null mutant, were rescued by the chimeric CESA5, and became comparable to the wild type (WT) and prc1-1 complemented by WT CESA6 in regard to plant growth, cellulose content, cellulose microfibril organization, CSC dynamics and subcellular localization. Bimolecular fluorescence complementation assays were employed to evaluate pairwise interactions between the N-terminal regions of CESA1, CESA3, CESA5, CESA6 and the chimeric CESA5. We verified that the chimeric CESA5 explicitly interacted with all the other CESA partners, comparable to CESA6, whereas interaction between CESA5 with itself was significantly weaker than that of all other CESA pairs. Our findings suggest that the homodimerization of CESA6 through its N-terminal zinc finger is critical in defining its functional properties, and possibly determines its intrinsic roles in facilitating higher-order structures in CSCs.

Direct Measurement of Plant Cellulose Microfibril and Bundles in Native Cell Walls.

Plants use rigid cellulose together with non-cellulosic matrix polymers to build cell walls. Cellulose microfibrils comprise linear ß(1,4)-glucan chains packed through inter- and intra-chain hydrogen-bonding networks and van der Waals forces. Due to its small size, the number of glucan chains and their arrangement in a microfibril remains elusive. Here we used atomic force microscopy (AFM) to directly image primary cell walls (PCWs) and secondary cell walls (SCWs) from fresh tissues of maize (Zea mays) under near-native conditions. By analyzing cellulose structure in different types of cell walls, we were able to measure the individual microfibrils in elongated PCWs at the sub-nanometer scale. The dimension of the microfibril was measured at 3.68 ± 0.13 nm in width and 2.25 ± 0.10 nm in height. By superimposing multiple AFM height profiles of these microfibrils, the overlay area representing the cross-section was estimated at 5.6 ± 0.4 nm2, which fitted well to an 18-chain model packed as six sheets with 234432 conformation. Interestingly we found in PCW, all these individual microfibrils could be traced back to a bundle in larger imaging area, suggesting cellulose are synthesized as large bundles in PCWs, and then split during cell expansion or elongation. In SCWs where cell growth has ceased we observed nearly-parallel twined or individual microfibrils that appeared to be embedded separately in the matrix polymers without the splitting effect, indicating different mechanisms of cellulose biosynthesis in PCW and SCW. The sub-nanometer structure of the microfibril presented here was measured exclusively from elongated PCWs, further study is required to verify if it represents the inherent structure synthesized by the cellulose synthase complex in PCWs and SCWs.

A mutation in the catalytic domain of cellulose synthase 6 halts its transport to the Golgi apparatus.

Cellulose microfibrils, which form the mechanical framework of the plant cell wall, are synthesized by the cellulose synthase complex in the plasma membrane. Here, we introduced point mutations into the catalytic domain of cellulose synthase 6 (CESA6) in Arabidopsis to produce enhanced yellow fluorescent protein (EYFP)-tagged CESA6D395N, CESA6Q823E, and CESA6D395N+Q823E, which were exogenously produced in a cesa6 null mutant, prc1-1. Comparison of these mutants in terms of plant phenotype, cellulose content, cellulose synthase complex dynamics, and organization of cellulose microfibrils showed that prc1-1 expressing EYFP:CESA6D395N or CESA6D395N+Q823E was nearly the same as prc1-1, whereas prc1-1 expressing EYFP:CESA6Q823E was almost identical to wild type and prc1-1 expressing EYFP:WT CESA6, indicating that CESA6D395N and CESA6D395N+Q823E do not function in cellulose synthesis, while CESA6Q823E is still functionally active. Total internal reflection fluorescence microscopy and confocal microscopy were used to monitor the subcellular localization of these proteins. We found that EYFP:CESA6D395N and EYFP:CESA6D395N+Q823E were absent from subcellular regions containing the Golgi and the plasma membrane, and they appeared to be retained in the endoplasmic reticulum. By contrast, EYFP:CESA6Q823E had a normal localization pattern, like that of wild-type EYFP:CESA6. Our results demonstrate that the D395N mutation in CESA6 interrupts its normal transport to the Golgi and its eventual participation in cellulose synthase complex assembly.

Identification and Characterization of Five Cold Stress-Related Rhododendron Dehydrin Genes: Spotlight on a FSK-Type Dehydrin With Multiple F-Segments.

Dehydrins are a family of plant proteins that accumulate in response to dehydration stresses, such as low temperature, drought, high salinity, or during seed maturation. We have previously constructed cDNA libraries from Rhododendron catawbiense leaves of naturally non-acclimated (NA; leaf LT50, temperature that results in 50% injury of maximum, approximately -7°C) and cold-acclimated (CA; leaf LT50 approximately -50°C) plants and analyzed expressed sequence tags (ESTs). Five ESTs were identified as dehydrin genes. Their full-length cDNA sequences were obtained and designated as RcDhn 1-5. To explore their functionality vis-à-vis winter hardiness, their seasonal expression kinetics was studied at two levels. Firstly, in leaves of R. catawbiense collected from the NA, CA, and de-acclimated (DA) plants corresponding to summer, winter and spring, respectively. Secondly, in leaves collected monthly from August through February, which progressively increased freezing tolerance from summer through mid-winter. The expression pattern data indicated that RcDhn 1-5 had 6- to 15-fold up-regulation during the cold acclimation process, followed by substantial down-regulation during deacclimation (even back to NA levels for some). Interestingly, our data shows RcDhn 5 contains a histidine-rich motif near N-terminus, a characteristic of metal-binding dehydrins. Equally important, RcDhn 2 contains a consensus 18 amino acid sequence (i.e., ETKDRGLFDFLGKKEEEE) near the N-terminus, with two additional copies upstream, and it is the most acidic (pI of 4.8) among the five RcDhns found. The core of this consensus 18 amino acid sequence is a 11-residue amino acid sequence (DRGLFDFLGKK), recently designated in the literature as the F-segment (based on the pair of hydrophobic F residues it contains). Furthermore, the 208 orthologs of F-segment-containing RcDhn 2 were identified across a broad range of species in GenBank database. This study expands our knowledge about the types of F-segment from the literature-reported single F-segment dehydrins (FSKn) to two or three F-segment dehydrins: Camelina sativa dehydrin ERD14 as F2S2Kn type; and RcDhn 2 as F3SKn type identified here. Our results also indicate some consensus amino acid sequences flanking the core F-segment in dehydrins. Implications for these cold-responsive RcDhn genes in future genetic engineering efforts to improve plant cold hardiness are discussed.

Real-time imaging reveals that lytic polysaccharide monooxygenase promotes cellulase activity by increasing cellulose accessibility.

BACKGROUND: The high cost of enzymes is one of the key technical barriers that must be overcome to realize the economical production of biofuels and biomaterials from biomass. Supplementation of enzyme cocktails with lytic polysaccharide monooxygenase (LPMO) can increase the efficiency of these cellulase mixtures for biomass conversion. The previous studies have revealed that LPMOs cleave polysaccharide chains by oxidization of the C1 and/or C4 carbons of the monomeric units. However, how LPMOs enhance enzymatic degradation of lignocellulose is still poorly understood. RESULTS: In this study, we combined enzymatic assays and real-time imaging using atomic force microscopy (AFM) to study the molecular interactions of an LPMO [TrAA9A, formerly known as TrCel61A) from Trichoderma reesei] and a cellobiohydrolase I (TlCel7A from T. longibrachiatum) with bacterial microcrystalline cellulose (BMCC) as a substrate. Cellulose conversion by TlCel7A alone was enhanced from 46 to 54% by the addition of TrAA9A. Conversion by a mixture of TlCel7A, endoglucanase, and ß-glucosidase was increased from 79 to 87% using pretreated BMCC with TrAA9A for 72 h. AFM imaging demonstrated that individual TrAA9A molecules exhibited intermittent random movement along, across, and penetrating into the ribbon-like microfibril structure of BMCC, which was concomitant with the release of a small amount of oxidized sugars and the splitting of large cellulose ribbons into fibrils with smaller diameters. The dividing effect of the cellulose microfibril occurred more rapidly when TrAA9A and TlCel7A were added together compared to TrAA9A alone; TlCel7A alone caused no separation. CONCLUSIONS: TrAA9A increases the accessible surface area of BMCC by separating large cellulose ribbons, and thereby enhances cellulose hydrolysis yield. By providing the first direct observation of LPMO action on a cellulosic substrate, this study sheds new light on the mechanisms by which LPMO enhances biomass conversion.

Co-Compartmentation of Terpene Biosynthesis and Storage via Synthetic Droplet.

Traditional bioproduct engineering focuses on pathway optimization, yet is often complicated by product inhibition, downstream consumption, and the toxicity of certain products. Here, we present the co-compartmentation of biosynthesis and storage via a synthetic droplet as an effective new strategy to improve the bioproduct yield, with squalene as a model compound. A hydrophobic protein was designed and introduced into the tobacco chloroplast to generate a synthetic droplet for terpene storage. Simultaneously, squalene biosynthesis enzymes were introduced to chloroplasts together with the droplet-forming protein to co-compartmentalize the biosynthesis and storage of squalene. The strategy has enabled a record yield of squalene at 2.6 mg/g fresh weight without compromising plant growth. Confocal fluorescent microscopy imaging, stimulated Raman scattering microscopy, and droplet composition analysis confirmed the formation of synthetic storage droplet in chloroplast. The co-compartmentation of synthetic storage droplet with a targeted metabolic pathway engineering represents a new strategy for enhancing bioproduct yield.

Three AtCesA6-like members enhance biomass production by distinctively promoting cell growth in Arabidopsis.

Cellulose is an abundant biopolymer and a prominent constituent of plant cell walls. Cellulose is also a central component to plant morphogenesis and contributes the bulk of a plant's biomass. While cellulose synthase (CesA) genes were identified over two decades ago, genetic manipulation of this family to enhance cellulose production has remained difficult. In this study, we show that increasing the expression levels of the three primary cell wall AtCesA6-like genes (AtCesA2, AtCesA5, AtCesA6), but not AtCesA3, AtCesA9 or secondary cell wall AtCesA7, can promote the expression of major primary wall CesA genes to accelerate primary wall CesA complex (cellulose synthase complexes, CSCs) particle movement for acquiring long microfibrils and consequently increasing cellulose production in Arabidopsis transgenic lines, as compared with wild-type. The overexpression transgenic lines displayed changes in expression of genes related to cell growth and proliferation, perhaps explaining the enhanced growth of the transgenic seedlings. Notably, overexpression of the three AtCesA6-like genes also enhanced secondary cell wall deposition that led to improved mechanical strength and higher biomass production in transgenic mature plants. Hence, we propose that overexpression of certain AtCesA genes can provide a biotechnological approach to increase cellulose synthesis and biomass accumulation in transgenic plants.

Visualizing chemical functionality in plant cell walls.

Understanding plant cell wall cross-linking chemistry and polymeric architecture is key to the efficient utilization of biomass in all prospects from rational genetic modification to downstream chemical and biological conversion to produce fuels and value chemicals. In fact, the bulk properties of cell wall recalcitrance are collectively determined by its chemical features over a wide range of length scales from tissue, cellular to polymeric architectures. Microscopic visualization of cell walls from the nanometer to the micrometer scale offers an in situ approach to study their chemical functionality considering its spatial and chemical complexity, particularly the capabilities of characterizing biomass non-destructively and in real-time during conversion processes. Microscopic characterization has revealed heterogeneity in the distribution of chemical features, which would otherwise be hidden in bulk analysis. Key microscopic features include cell wall type, wall layering, and wall composition-especially cellulose and lignin distributions. Microscopic tools, such as atomic force microscopy, stimulated Raman scattering microscopy, and fluorescence microscopy, have been applied to investigations of cell wall structure and chemistry from the native wall to wall treated by thermal chemical pretreatment and enzymatic hydrolysis. While advancing our current understanding of plant cell wall recalcitrance and deconstruction, microscopic tools with improved spatial resolution will steadily enhance our fundamental understanding of cell wall function.

Directed plant cell-wall accumulation of iron: embedding co-catalyst for efficient biomass conversion.

BACKGROUND: Plant lignocellulosic biomass is an abundant, renewable feedstock for the production of biobased fuels and chemicals. Previously, we showed that iron can act as a co-catalyst to improve the deconstruction of lignocellulosic biomass. However, directly adding iron catalysts into biomass prior to pretreatment is diffusion limited, and increases the cost of biorefinery operations. Recently, we developed a new strategy for expressing iron-storage protein ferritin intracellularly to accumulate iron as a catalyst for the downstream deconstruction of lignocellulosic biomass. In this study, we extend this approach by fusing the heterologous ferritin gene with a signal peptide for secretion into Arabidopsis cell walls (referred to here as FerEX). RESULTS: The transgenic Arabidopsis plants. FerEX. accumulated iron under both normal and iron-fertilized growth conditions; under the latter (iron-fertilized) condition, FerEX transgenic plants showed an increase in plant height and dry weight by 12 and 18 %, respectively, compared with the empty vector control plants. The SDS- and native-PAGE separation of cell-wall protein extracts followed by Western blot analyses confirmed the extracellular expression of ferritin in FerEX plants. Meanwhile, Perls' Prussian blue staining and X-ray fluorescence microscopy (XFM) maps revealed iron depositions in both the secondary and compound middle lamellae cell-wall layers, as well as in some of the corner compound middle lamella in FerEX. Remarkably, their harvested biomasses showed enhanced pretreatability and digestibility, releasing, respectively, 21 % more glucose and 34 % more xylose than the empty vector control plants. These values are significantly higher than those of our recently obtained ferritin intracellularly expressed plants. CONCLUSIONS: This study demonstrated that extracellular expression of ferritin in Arabidopsis can produce plants with increased growth and iron accumulation, and reduced thermal and enzymatic recalcitrance. The results are attributed to the intimate colocation of the iron co-catalyst and the cellulose and hemicellulose within the plant cell-wall region, supporting the genetic modification strategy for incorporating conversion catalysts into energy crops prior to harvesting or processing at the biorefinery.

Burkholderia phytofirmans Inoculation-Induced Changes on the Shoot Cell Anatomy and Iron Accumulation Reveal Novel Components of Arabidopsis-Endophyte Interaction that Can Benefit Downstream Biomass Deconstruction.

It is known that plant growth promoting bacteria (PGPB) elicit positive effects on plant growth and biomass yield. However, the actual mechanism behind the plant-PGPB interaction is poorly understood, and the literature is scarce regarding the thermochemical pretreatability and enzymatic degradability of biomass derived from PGPB-inoculated plants. Most recent transcriptional analyses of PGPB strain Burkholderia phytofirmans PsJN inoculating potato in literature and Arabidopsis in our present study have revealed the expression of genes for ferritin and the biosynthesis and transport of siderophores (i.e., the molecules with high affinity for iron), respectively. The expression of such genes in the shoots of PsJN-inoculated plants prompted us to propose that PsJN-inoculation can improve the host plant's iron uptake and accumulation, which facilitates the downstream plant biomass pretreatment and conversion to simple sugars. In this study, we employed B. phytofirmans PsJN to inoculate the Arabidopsis thaliana plants, and conducted the first investigation for its effects on the biomass yield, the anatomical organization of stems, the iron accumulation, and the pretreatment and enzymatic hydrolysis of harvested biomass. The results showed that the strain PsJN stimulated plant growth in the earlier period of plant development and enlarged the cell size of stem piths, and it also indeed enhanced the essential metals uptake and accumulation in host plants. Moreover, we found that the PsJN-inoculated plant biomass released more glucose and xylose after hot water pretreatment and subsequent co-saccharification, which provided a novel insight into development of lignocellulosic biofuels from renewable biomass resources.

In situ micro-spectroscopic investigation of lignin in poplar cell walls pretreated by maleic acid.

BACKGROUND: In higher plant cells, lignin provides necessary physical support for plant growth and resistance to attack by microorganisms. For the same reason, lignin is considered to be a major impediment to the process of deconstructing biomass to simple sugars by hydrolytic enzymes. The in situ variation of lignin in plant cell walls is important for better understanding of the roles lignin play in biomass recalcitrance. RESULTS: A micro-spectroscopic approach combining stimulated Raman scattering microscopy and fluorescence lifetime imaging microscopy was employed to probe the physiochemical structure of lignin in poplar tracheid cell walls. Two forms of lignins were identified: loosely packed lignin, which had a long (4 ns) fluorescence lifetime and existed primarily in the secondary wall layers; and dense lignin, which had a short (0.5-1 ns) fluorescence lifetime and was present in all wall layers, including the cell corners, compound middle lamellae, and secondary wall. At low maleic acid concentration (0.025 and 0.05 M) pretreatment conditions, some of the dense lignin was modified to become more loosely packed. High acid concentration removed both dense and loosely packed lignins. These modified lignins reformed to make lignin-carbohydrate complex droplets containing either dense or loosely packed lignin (mostly from secondary walls) and were commonly observed on the cell wall surface. CONCLUSIONS: We have identified dense and loosely packed lignins in plant cell walls. During maleic acid pretreatment, both dense lignin droplets and loosely packed lignin droplets were formed. Maleic acid pretreatment more effectively removes loosely packed lignin in secondary walls which increases enzyme accessibility for digestion.

Identifying the ionically bound cell wall and intracellular glycoside hydrolases in late growth stage Arabidopsis stems: implications for the genetic engineering of bioenergy crops.

Identifying the cell wall-ionically bound glycoside hydrolases (GHs) in Arabidopsis stems is important for understanding the regulation of cell wall integrity. For cell wall proteomics studies, the preparation of clean cell wall fractions is a challenge since cell walls constitute an open compartment, which is more likely to contain a mixture of intracellular and extracellular proteins due to cell leakage at the late growth stage. Here, we utilize a CaCl2-extraction procedure to isolate non-structural proteins from Arabidopsis whole stems, followed by the in-solution and in-gel digestion methods coupled with Nano-LC-MS/MS, bioinformatics and literature analyses. This has led to the identification of 75 proteins identified using the in-solution method and 236 proteins identified by the in-gel method, among which about 10% of proteins predicted to be secreted. Together, eight cell wall proteins, namely AT1G75040, AT5G26000, AT3G57260, AT4G21650, AT3G52960, AT3G49120, AT5G49360, and AT3G14067, were identified by the in-solution method; among them, three were the GHs (AT5G26000, myrosinase 1, GH1; AT3G57260, ß-1,3-glucanase 2, GH17; AT5G49360, bifunctional XYL 1/α-L-arabinofuranosidase, GH3). Moreover, four more GHs: AT4G30270 (xyloglucan endotransferase, GH16), AT1G68560 (bifunctional α-l-arabinofuranosidase/XYL, GH31), AT1G12240 (invertase, GH32) and AT2G28470 (ß-galactosidase 8, GH35), were identified by the in-gel solution method only. Notably, more than half of above identified GHs are xylan- or hemicellulose-modifying enzymes, and will likely have an impact on cellulose accessibility, which is a critical factor for downstream enzymatic hydrolysis of plant tissues for biofuels production. The implications of these cell wall proteins identified at the late growth stage for the genetic engineering of bioenergy crops are discussed.

Pinoresinol reductase 1 impacts lignin distribution during secondary cell wall biosynthesis in Arabidopsis.

Pinoresinol reductase (PrR) catalyzes the conversion of the lignan (-)-pinoresinol to (-)-lariciresinol in Arabidopsis thaliana, where it is encoded by two genes, PrR1 and PrR2, that appear to act redundantly. PrR1 is highly expressed in lignified inflorescence stem tissue, whereas PrR2 expression is barely detectable in stems. Co-expression analysis has indicated that PrR1 is co-expressed with many characterized genes involved in secondary cell wall biosynthesis, whereas PrR2 expression clusters with a different set of genes. The promoter of the PrR1 gene is regulated by the secondary cell wall related transcription factors SND1 and MYB46. The loss-of-function mutant of PrR1 shows, in addition to elevated levels of pinoresinol, significantly decreased lignin content and a slightly altered lignin structure with lower abundance of cinnamyl alcohol end groups. Stimulated Raman scattering (SRS) microscopy analysis indicated that the lignin content of the prr1-1 loss-of-function mutant is similar to that of wild-type plants in xylem cells, which exhibit a normal phenotype, but is reduced in the fiber cells. Together, these data suggest an association of the lignan biosynthetic enzyme encoded by PrR1 with secondary cell wall biosynthesis in fiber cells.

Lignin plays a negative role in the biochemical process for producing lignocellulosic biofuels.

A biochemical platform holds the most promising route toward lignocellulosic biofuels, in which polysaccharides are hydrolyzed by cellulase enzymes into simple sugars and fermented to ethanol by microbes. However, these polysaccharides are cross-linked in the plant cell walls with the hydrophobic network of lignin that physically impedes enzymatic deconstruction. A thermochemical pretreatment process is often required to remove or delocalize lignin, which may also generate inhibitors that hamper enzymatic hydrolysis and fermentation. Here we review recent advances in understanding lignin structure in the plant cell walls and the negative roles of lignin in the processes of converting biomass to biofuels. Perspectives and future directions to improve the biomass conversion process are also discussed.

Comparison of transcriptional profiles of Clostridium thermocellum grown on cellobiose and pretreated yellow poplar using RNA-Seq.

The anaerobic, thermophilic bacterium, Clostridium thermocellum, secretes multi-protein enzyme complexes, termed cellulosomes, which synergistically interact with the microbial cell surface and efficiently disassemble plant cell wall biomass. C. thermocellum has also been considered a potential consolidated bioprocessing (CBP) organism due to its ability to produce the biofuel products, hydrogen, and ethanol. We found that C. thermocellum fermentation of pretreated yellow poplar (PYP) produced 30 and 39% of ethanol and hydrogen product concentrations, respectively, compared to fermentation of cellobiose. RNA-seq was used to analyze the transcriptional profiles of these cells. The PYP-grown cells taken for analysis at the late stationary phase showed 1211 genes up-regulated and 314 down-regulated by more than two-fold compared to the cellobiose-grown cells. These affected genes cover a broad spectrum of specific functional categories. The transcriptional analysis was further validated by sub-proteomics data taken from the literature; as well as by quantitative reverse transcription-PCR (qRT-PCR) analyses of selected genes. Specifically, 47 cellulosomal protein-encoding genes, genes for 4 pairs of SigI-RsgI for polysaccharide sensing, 7 cellodextrin ABC transporter genes, and a set of NAD(P)H hydogenase and alcohol dehydrogenase genes were up-regulated for cells growing on PYP compared to cellobiose. These genes could be potential candidates for future studies aimed at gaining insight into the regulatory mechanism of this organism as well as for improvement of C. thermocellum in its role as a CBP organism.

Agave proves to be a low recalcitrant lignocellulosic feedstock for biofuels production on semi-arid lands.

BACKGROUND: Agave, which is well known for tequila and other liquor production in Mexico, has recently gained attention because of its attractive potential to launch sustainable bioenergy feedstock solutions for semi-arid and arid lands. It was previously found that agave cell walls contain low lignin and relatively diverse non-cellulosic polysaccharides, suggesting unique recalcitrant features when compared to conventional C4 and C3 plants. RESULTS: Here, we report sugar release data from fungal enzymatic hydrolysis of non-pretreated and hydrothermally pretreated biomass that shows agave to be much less recalcitrant to deconstruction than poplar or switchgrass. In fact, non-pretreated agave has a sugar release five to eight times greater than that of poplar wood and switchgrass . Meanwhile, state of the art techniques including glycome profiling, nuclear magnetic resonance (NMR), Simon's Stain, confocal laser scanning microscopy and so forth, were applied to measure interactions of non-cellulosic wall components, cell wall hydrophilicity, and enzyme accessibility to identify key structural features that make agave cell walls less resistant to biological deconstruction when compared to poplar and switchgrass. CONCLUSIONS: This study systematically evaluated the recalcitrant features of agave plants towards biofuels applications. The results show that not only does agave present great promise for feeding biorefineries on semi-arid and arid lands, but also show the value of studying agave's low recalcitrance for developments in improving cellulosic energy crops.

Improving activity of minicellulosomes by integration of intra- and intermolecular synergies.

BACKGROUND: Complete hydrolysis of cellulose to glucose requires the synergistic action of three general types of glycoside hydrolases; endoglucanases, exoglucanases, and cellobiases. Cellulases that are found in Nature vary considerably in their modular diversity and architecture. They include: non-complexed enzymes with single catalytic domains, independent single peptide chains incorporating multiple catalytic modules, and complexed, scaffolded structures, such as the cellulosome. The discovery of the latter two enzyme architectures has led to a generally held hypothesis that these systems take advantage of intramolecular and intermolecular proximity synergies, respectively, to enhance cellulose degradation. We use domain engineering to exploit both of these concepts to improve cellulase activity relative to the activity of mixtures of the separate catalytic domains. RESULTS: We show that engineered minicellulosomes can achieve high levels of cellulose conversion on crystalline cellulose by taking advantage of three types of synergism; (1) a complementary synergy produced by interaction of endo- and exo-cellulases, (2) an intramolecular synergy of multiple catalytic modules in a single gene product (this type of synergism being introduced for the first time to minicellulosomes targeting crystalline cellulose), and (3) an intermolecular proximity synergy from the assembly of these cellulases into larger multi-molecular structures called minicellulosomes. The binary minicellulosome constructed in this study consists of an artificial multicatalytic cellulase (CBM4-Ig-GH9-X11-X12-GH8-Doc) and one cellulase with a single catalytic domain (a modified Cel48S with the structure CBM4-Ig-GH48-Doc), connected by a non-catalytic scaffoldin protein. The high level endo-exo synergy and intramolecular synergies within the artificial multifunctional cellulase have been combined with an additional proximity-dependent synergy produced by incorporation into a minicellulosome demonstrating high conversion of crystalline cellulose (Avicel). Our minicellulosome is the first engineered enzyme system confirmed by test to be capable of both operating at temperatures as high as 60°C and converting over 60% of crystalline cellulose to fermentable sugars. CONCLUSION: When compared to previously reported minicellulosomes assembled from cellulases containing only one catalytic module each, our novel minicellulosome demonstrates a method for substantial reduction in the number of peptide chains required, permitting improved heterologous expression of minicellulosomes in microbial hosts. In addition, it has been shown to be capable of substantial conversion of actual crystalline cellulose, as well as of the less-well-ordered and more easily digestible fraction of nominally crystalline cellulose.