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The lignin biosynthetic pathway is highly conserved in angiosperms, yet pathway manipulations give rise to a variety of taxon-specific outcomes. Knockout of lignin-associated 4-coumarate:CoA ligases (4CLs) in herbaceous species mainly reduces guaiacyl (G) lignin and enhances cell wall saccharification. Here we show that CRISPR-knockout of 4CL1 in poplar (Populus tremula × alba) preferentially reduced syringyl (S) lignin, with negligible effects on biomass recalcitrance. Concordant with reduced S-lignin was downregulation of ferulate 5-hydroxylases (F5Hs). Lignification was largely sustained by 4CL5, a low-affinity paralog of 4CL1 typically with only minor xylem expression or activity. Levels of caffeate, the preferred substrate of 4CL5, increased in line with significant upregulation of caffeoyl shikimate esterase1 Upregulation of caffeoyl-CoA O-methyltransferase1 and downregulation of F5Hs are consistent with preferential funneling of 4CL5 products toward G-lignin biosynthesis at the expense of S-lignin. Thus, transcriptional and metabolic adaptations to 4CL1-knockout appear to have enabled 4CL5 catalysis at a level sufficient to sustain lignification. Finally, genes involved in sulfur assimilation, the glutathione-ascorbate cycle, and various antioxidant systems were upregulated in the mutants, suggesting cascading responses to perturbed thioesterification in lignin biosynthesis.
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Lignina/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Populus/metabolismo , Xilema/metabolismo , Carboxiliases/genética , Carboxiliases/metabolismo , Catálise , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/genética , Xilema/genéticaRESUMO
[This corrects the article DOI: 10.1186/s13068-019-1353-7.].
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Background: The recalcitrance of cellulosic biomass is widely recognized as a key barrier to cost-effective biological processing to fuels and chemicals, but the relative impacts of physical, chemical and genetic interventions to improve biomass processing singly and in combination have yet to be evaluated systematically. Solubilization of plant cell walls can be enhanced by non-biological augmentation including physical cotreatment and thermochemical pretreatment, the choice of biocatalyst, the choice of plant feedstock, genetic engineering of plants, and choosing feedstocks that are less recalcitrant natural variants. A two-tiered combinatoric investigation of lignocellulosic biomass deconstruction was undertaken with three biocatalysts (Clostridium thermocellum, Caldicellulosiruptor bescii, Novozymes Cellic® Ctec2 and Htec2), three transgenic switchgrass plant lines (COMT, MYB4, GAUT4) and their respective nontransgenic controls, two Populus natural variants, and augmentation of biological attack using either mechanical cotreatment or cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment. Results: In the absence of augmentation and under the conditions tested, increased total carbohydrate solubilization (TCS) was observed for 8 of the 9 combinations of switchgrass modifications and biocatalysts tested, and statistically significant for five of the combinations. Our results indicate that recalcitrance is not a trait determined by the feedstock only, but instead is coequally determined by the choice of biocatalyst. TCS with C. thermocellum was significantly higher than with the other two biocatalysts. Both CELF pretreatment and cotreatment via continuous ball milling enabled TCS in excess of 90%. Conclusion: Based on our results as well as literature studies, it appears that some form of non-biological augmentation will likely be necessary for the foreseeable future to achieve high TCS for most cellulosic feedstocks. However, our results show that this need not necessarily involve thermochemical processing, and need not necessarily occur prior to biological conversion. Under the conditions tested, the relative magnitude of TCS increase was augmentation > biocatalyst choice > plant choice > plant modification > plant natural variants. In the presence of augmentation, plant modification, plant natural variation, and plant choice exhibited a small, statistically non-significant impact on TCS.
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A multi-omics quantitative integrative analysis of lignin biosynthesis can advance the strategic engineering of wood for timber, pulp, and biofuels. Lignin is polymerized from three monomers (monolignols) produced by a grid-like pathway. The pathway in wood formation of Populus trichocarpa has at least 21 genes, encoding enzymes that mediate 37 reactions on 24 metabolites, leading to lignin and affecting wood properties. We perturb these 21 pathway genes and integrate transcriptomic, proteomic, fluxomic and phenomic data from 221 lines selected from ~2000 transgenics (6-month-old). The integrative analysis estimates how changing expression of pathway gene or gene combination affects protein abundance, metabolic-flux, metabolite concentrations, and 25 wood traits, including lignin, tree-growth, density, strength, and saccharification. The analysis then predicts improvements in any of these 25 traits individually or in combinations, through engineering expression of specific monolignol genes. The analysis may lead to greater understanding of other pathways for improved growth and adaptation.
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Lignina/biossíntese , Lignina/genética , Populus/genética , Madeira/química , Madeira/fisiologia , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/genética , Populus/metabolismo , Transcriptoma/genética , Árvores/genética , Árvores/metabolismo , Xilema/metabolismoRESUMO
Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.
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Biocombustíveis , Parede Celular/genética , Glucuronosiltransferase/genética , Pectinas/biossíntese , Biomassa , Boro/metabolismo , Cálcio/metabolismo , Parede Celular/enzimologia , Parede Celular/metabolismo , Produtos Agrícolas , Glucuronosiltransferase/química , Panicum/enzimologia , Panicum/genética , Pectinas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Populus/enzimologia , Populus/genética , Açúcares/metabolismoRESUMO
As a consequence of their remarkable adaptability, fast growth, and superior wood properties, eucalypt tree plantations have emerged as key renewable feedstocks (over 20 million ha globally) for the production of pulp, paper, bioenergy, and other lignocellulosic products. However, most biomass properties such as growth, wood density, and wood chemistry are complex traits that are hard to improve in long-lived perennials. Systems genetics, a process of harnessing multiple levels of component trait information (e.g., transcript, protein, and metabolite variation) in populations that vary in complex traits, has proven effective for dissecting the genetics and biology of such traits. We have applied a network-based data integration (NBDI) method for a systems-level analysis of genes, processes and pathways underlying biomass and bioenergy-related traits using a segregating Eucalyptus hybrid population. We show that the integrative approach can link biologically meaningful sets of genes to complex traits and at the same time reveal the molecular basis of trait variation. Gene sets identified for related woody biomass traits were found to share regulatory loci, cluster in network neighborhoods, and exhibit enrichment for molecular functions such as xylan metabolism and cell wall development. These findings offer a framework for identifying the molecular underpinnings of complex biomass and bioprocessing-related traits. A more thorough understanding of the molecular basis of plant biomass traits should provide additional opportunities for the establishment of a sustainable bio-based economy.
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Biomassa , Eucalyptus/metabolismo , Redes Reguladoras de Genes , Genes de Plantas , Lignina/metabolismo , Redes e Vias Metabólicas/genética , Modelos Genéticos , Carbono/metabolismo , Parede Celular/metabolismo , Mapeamento Cromossômico , Cruzamentos Genéticos , Eucalyptus/genética , Eucalyptus/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Hibridização Genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Locos de Características Quantitativas , Madeira/metabolismoRESUMO
BACKGROUND: The native recalcitrance of plants hinders the biomass conversion process using current biorefinery techniques. Down-regulation of the caffeic acid O-methyltransferase (COMT) gene in the lignin biosynthesis pathway of switchgrass reduced the thermochemical and biochemical conversion recalcitrance of biomass. Due to potential environmental influences on lignin biosynthesis and deposition, studying the consequences of physicochemical changes in field-grown plants without pretreatment is essential to evaluate the performance of lignin-altered plants. We determined the chemical composition, cellulose crystallinity and the degree of its polymerization, molecular weight of hemicellulose, and cellulose accessibility of cell walls in order to better understand the fundamental features of why biomass is recalcitrant to conversion without pretreatment. The most important is to investigate whether traits and features are stable in the dynamics of field environmental effects over multiple years. RESULTS: Field-grown COMT down-regulated plants maintained both reduced cell wall recalcitrance and lignin content compared with the non-transgenic controls for at least 3 seasons. The transgenic switchgrass yielded 35-84% higher total sugar release (enzymatic digestibility or saccharification) from a 72-h enzymatic hydrolysis without pretreatment and also had a 25-32% increase in enzymatic sugar release after hydrothermal pretreatment. The COMT-silenced switchgrass lines had consistently lower lignin content, e.g., 12 and 14% reduction for year 2 and year 3 growing season, respectively, than the control plants. By contrast, the transgenic lines had 7-8% more xylan and galactan contents than the wild-type controls. Gel permeation chromatographic results revealed that the weight-average molecular weights of hemicellulose were 7-11% lower in the transgenic than in the control lines. In addition, we found that silencing of COMT in switchgrass led to 20-22% increased cellulose accessibility as measured by the Simons' stain protocol. No significant changes were observed on the arabinan and glucan contents, cellulose crystallinity, and cellulose degree of polymerization between the transgenic and control plants. With the 2-year comparative analysis, both the control and transgenic lines had significant increases in lignin and glucan contents and hemicellulose molecular weight across the growing seasons. CONCLUSIONS: The down-regulation of COMT in switchgrass resulting in a reduced lignin content and biomass recalcitrance is stable in a field-grown trial for at least three seasons. Among the determined affecting factors, the reduced biomass recalcitrance of the COMT-silenced switchgrass, grown in the field conditions for two and three seasons, was likely related to the decreased lignin content and increased biomass accessibility, whereas the cellulose crystallinity and degree of its polymerization and hemicellulose molecular weights did not contribute to the reduction of recalcitrance significantly. This finding suggests that lignin down-regulation in lignocellulosic feedstock confers improved saccharification that translates from greenhouse to field trial and that lignin content and biomass accessibility are two significant factors for developing a reduced recalcitrance feedstock by genetic modification.
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In an effort to better understand the biomass recalcitrance, six natural poplar variants were selected as feedstocks based on previous sugar release analysis. Compositional analysis and physicochemical characterizations of these poplars were performed and the correlations between these physicochemical properties and enzymatic hydrolysis yield were investigated. Gel permeation chromatography (GPC) and 13 Câ solid state NMR were used to determine the degree of polymerization (DP) and crystallinity index (CrI) of cellulose, and the results along with the sugar release study indicated that cellulose DP likely played a more important role in enzymatic hydrolysis. Simons' stain revealed that the accessible surface area of substrate significantly varied among these variants from 17.3 to 33.2â mg g-1biomass as reflected by dye adsorption, and cellulose accessibility was shown as one of the major factors governing substrates digestibility. HSQC and 31 Pâ NMR analysis detailed the structural features of poplar lignin variants. Overall, cellulose relevant factors appeared to have a stronger correlation with glucose release, if any, than lignin structural features. Lignin structural features, such as a phenolic hydroxyl group and the ratio of syringyl and guaiacyl (S/G), were found to have a more convincing impact on xylose release. Low lignin content, low cellulose DP, and high cellulose accessibility generally favor enzymatic hydrolysis; however, recalcitrance cannot be simply judged on any single substrate factor.
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Biocombustíveis , Biomassa , Populus/química , Biotecnologia , Celulose/química , Hidrólise , Lignina/química , Polimerização , Populus/crescimento & desenvolvimentoRESUMO
Plant laccases are thought to function in the oxidation of monolignols which leads to higher order lignin formation. Only a hand-full of laccases in plants have been functionally evaluated, and as such little is known about the breadth of their impact on cell wall chemistry or structure. Here, we describe a previously uncharacterized laccase from Populus, encoded by locus Potri.008G064000, whose reduced expression resulted in transgenic Populus trees with changes in syringyl/guaiacyl ratios as well as altered sugar release phenotypes. These phenotypes are consistent with plant biomass exhibiting reduced recalcitrance. Interestingly, the transgene effect on recalcitrance is dependent on a mild pretreatment prior to chemical extraction of sugars. Metabolite profiling suggests the transgene modulates phenolics that are associated with the cell wall structure. We propose that this particular laccase has a range of functions related to oxidation of phenolics and conjugation of flavonoids that interact with lignin in the cell wall.
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Parede Celular/química , Lacase/metabolismo , Plantas Geneticamente Modificadas/enzimologia , Populus/enzimologia , Populus/genética , Parede Celular/enzimologia , Parede Celular/genética , Regulação da Expressão Gênica de Plantas/genética , Lacase/genética , Lignina/metabolismo , Plantas Geneticamente Modificadas/genética , Xilose/metabolismoRESUMO
BACKGROUND: Feedstock recalcitrance is the most important barrier impeding cost-effective production of cellulosic biofuels. Pioneer commercial cellulosic ethanol facilities employ thermochemical pretreatment and addition of fungal cellulase, reflecting the main research emphasis in the field. However, it has been suggested that it may be possible to process cellulosic biomass without thermochemical pretreatment using thermophilic, cellulolytic bacteria. To further explore this idea, we examine the ability of various biocatalysts to solubilize autoclaved but otherwise unpretreated cellulosic biomass under controlled but not industrial conditions. RESULTS: Carbohydrate solubilization of mid-season harvested switchgrass after 5 days ranged from 24 % for Caldicellulosiruptor bescii to 65 % for Clostridium thermocellum, with intermediate values for a thermophilic horse manure enrichment, Clostridium clariflavum, Clostridium cellulolyticum, and simultaneous saccharification and fermentation (SSF) featuring a fungal cellulase cocktail and yeast. Under a variety of conditions, solubilization yields were about twice as high for C. thermocellum compared to fungal cellulase. Solubilization of mid-season harvested switchgrass was about twice that of senescent switchgrass. Lower yields and greater dependence on particle size were observed for Populus as compared to switchgrass. Trends observed from data drawn from six conversion systems and three substrates, including both time course and end-point data, were (1) equal fractional solubilization of glucan and xylan, (2) no biological solubilization of the non-carbohydrate fraction of biomass, and (3) higher solubilization for three of the four bacterial cultures tested as compared to the fungal cellulase system. Brief (5 min) ball milling of solids remaining after fermentation of senescent switchgrass by C. thermocellum nearly doubled carbohydrate solubilization upon reinnoculation as compared to a control without milling. Greater particle size reduction and solubilization were observed for milling of partially fermented solids than for unfermented solids. Physical disruption of cellulosic feedstocks after initiation of fermentation, termed cotreatment, warrants further study. CONCLUSIONS: While the ability to achieve significant solubilization of minimally pretreated switchgrass is widespread, a fivefold difference between the most and least effective biocatalyst-feedstock combinations was observed. Starting with nature's best biomass-solubilizing systems may enable a reduction in the amount of non-biological processing required, and in particular substitution of cotreatment for pretreatment.
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Ionic liquids have great potential in biological applications and biocatalysis, as some ionic liquids can stabilize proteins and enhance enzyme activity, while others have the opposite effect. However, on the molecular level, probing ionic liquid interactions with proteins, especially in solutions containing high concentration of ionic liquids, has been challenging. In the present work the 13C, 15N-enriched GB1 model protein was used to demonstrate applicability of high-resolution magic-angle-spinning (HR-MAS) NMR spectroscopy to investigate ionic liquid - protein interactions. Effect of an ionic liquid (1-butyl-3-methylimidazolium bromide, [C4-mim]Br) on GB1was studied over a wide range of the ionic liquid concentrations (0.6 to 3.5 M, which corresponds to 10%-60% v/v). Interactions between GB1 and [C4-mim]Br were observed from changes in the chemical shifts of the protein backbone as well as the changes in 15N ps-ns dynamics and rotational correlation times. Site-specific interactions between the protein and [C4-mim]Br were assigned using 3D methods under HR-MAS conditions. Thus, HR-MAS NMR is a viable tool that could aid in elucidation of the molecular mechanism of ionic liquid - protein interactions.
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The conversion of lignocellulosic biomass to fuels, chemicals, and other commodities has been explored as one possible pathway toward reductions in the use of non-renewable energy sources. In order to identify which plants, out of a diverse pool, have the desired chemical traits for downstream applications, attributes, such as cellulose and lignin content, or monomeric sugar release following an enzymatic saccharification, must be compared. The experimental and data analysis protocols of the standard methods of analysis can be time-consuming, thereby limiting the number of samples that can be measured. High-throughput (HTP) methods alleviate the shortcomings of the standard methods, and permit the rapid screening of available samples to isolate those possessing the desired traits. This study illustrates the HTP sugar release and pyrolysis-molecular beam mass spectrometry pipelines employed at the National Renewable Energy Lab. These pipelines have enabled the efficient assessment of thousands of plants while decreasing experimental time and costs through reductions in labor and consumables.
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Biomassa , Carboidratos/análise , Lignina/química , Carboidratos/química , Cromatografia Líquida de Alta Pressão/métodos , Glucose/análise , Ensaios de Triagem em Larga Escala/métodos , Plantas/química , Xilose/análiseRESUMO
BACKGROUND: Lignocellulosic materials provide an attractive replacement for food-based crops used to produce ethanol. Understanding the interactions within the cell wall is vital to overcome the highly recalcitrant nature of biomass. One factor imparting plant cell wall recalcitrance is lignin, which can be manipulated by making changes in the lignin biosynthetic pathway. In this study, eucalyptus down-regulated in expression of cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) or p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H, EC 1.14.13.36) were evaluated for cell wall composition and reduced recalcitrance. RESULTS: Eucalyptus trees with down-regulated C4H or C3'H expression displayed lowered overall lignin content. The control samples had an average of 29.6 %, the C3'H reduced lines had an average of 21.7 %, and the C4H reduced lines had an average of 18.9 % lignin from wet chemical analysis. The C3'H and C4H down-regulated lines had different lignin compositions with average S/G/H ratios of 48.5/33.2/18.3 for the C3'H reduced lines and 59.0/39.8/1.2 for the C4H reduced lines, compared to the control with 65.9/33.2/1.0. Both the C4H and C3'H down-regulated lines had reduced recalcitrance as indicated by increased sugar release as determined using enzymatic conversion assays utilizing both no pretreatment and a hot water pretreatment. CONCLUSIONS: Lowering lignin content rather than altering sinapyl alcohol/coniferyl alcohol/4-coumaryl alcohol ratios was found to have the largest impact on reducing recalcitrance of the transgenic eucalyptus variants. The development of lower recalcitrance trees opens up the possibility of using alternative pretreatment strategies in biomass conversion processes that can reduce processing costs.
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The creation of fuels, chemicals, and materials from plants can aid in replacing products fabricated from non-renewable energy sources. Before using biomass in downstream applications, it must be characterized to assess chemical traits, such as cellulose, lignin, or lignin monomer content, or the sugars released following an acid or enzymatic hydrolysis. The measurement of these traits allows researchers to gage the recalcitrance of the plants and develop efficient deconstruction strategies to maximize yields. Standard methods for assessing biomass phenotypes often have experimental protocols that limit their use for screening sizeable numbers of plant species. Raman spectroscopy, a non-destructive, non-invasive vibrational spectroscopy technique, is capable of providing qualitative, structural information and quantitative measurements. Applications of Raman spectroscopy have aided in alleviating the constraints of standard methods by coupling spectral data with multivariate analysis to construct models capable of predicting analytes. Hydrolysis and fermentation products, such as glucose and ethanol, can be quantified off-, at-, or on-line. Raman imaging has enabled researchers to develop a visual understanding of reactions, such as different pretreatment strategies, in real-time, while also providing integral chemical information. This review provides an overview of what Raman spectroscopy is, and how it has been applied to the analysis of whole lignocellulosic biomass, its derivatives, and downstream process monitoring.
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BACKGROUND: The inherent recalcitrance of woody bioenergy feedstocks is a major challenge for their use as a source of second-generation biofuel. Secondary cell walls that constitute the majority of hardwood biomass are rich in cellulose, xylan, and lignin. The interactions among these polymers prevent facile accessibility and deconstruction by enzymes and chemicals. Plant biomass that can with minimal pretreatment be degraded into sugars is required to produce renewable biofuels in a cost-effective manner. RESULTS: GAUT12/IRX8 is a putative glycosyltransferase proposed to be involved in secondary cell wall glucuronoxylan and/or pectin biosynthesis based on concomitant reductions of both xylan and the pectin homogalacturonan (HG) in Arabidopsis irx8 mutants. Two GAUT12 homologs exist in Populus trichocarpa, PtGAUT12.1 and PtGAUT12.2. Knockdown expression of both genes simultaneously has been shown to reduce xylan content in Populus wood. We tested the proposition that RNA interference (RNAi) downregulation of GAUT12.1 alone would lead to increased sugar release in Populus wood, that is, reduced recalcitrance, based on the hypothesis that GAUT12 synthesizes a wall structure required for deposition of xylan and that cell walls with less xylan and/or modified cell wall architecture would have reduced recalcitrance. Using an RNAi approach, we generated 11 Populus deltoides transgenic lines with 50 to 67% reduced PdGAUT12.1 transcript expression compared to wild type (WT) and vector controls. Ten of the eleven RNAi lines yielded 4 to 8% greater glucose release upon enzymatic saccharification than the controls. The PdGAUT12.1 knockdown (PdGAUT12.1-KD) lines also displayed 12 to 52% and 12 to 44% increased plant height and radial stem diameter, respectively, compared to the controls. Knockdown of PdGAUT12.1 resulted in a 25 to 47% reduction in galacturonic acid and 17 to 30% reduction in xylose without affecting total lignin content, revealing that in Populus wood as in Arabidopsis, GAUT12 affects both pectin and xylan formation. Analyses of the sugars present in sequential cell wall extracts revealed a reduction of glucuronoxylan and pectic HG and rhamnogalacturonan in extracts from PdGAUT12.1-KD lines. CONCLUSIONS: The results show that downregulation of GAUT12.1 leads to a reduction in a population of xylan and pectin during wood formation and to reduced recalcitrance, more easily extractable cell walls, and increased growth in Populus.
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Central carbon metabolism in cyanobacteria comprises the Calvin-Benson-Bassham (CBB) cycle, glycolysis, the pentose phosphate (PP) pathway and the tricarboxylic acid (TCA) cycle. Redundancy in this complex metabolic network renders the rational engineering of cyanobacterial metabolism for the generation of biomass, biofuels and chemicals a challenge. Here we report the presence of a functional phosphoketolase pathway, which splits xylulose-5-phosphate (or fructose-6-phosphate) to acetate precursor acetyl phosphate, in an engineered strain of the model cyanobacterium Synechocystis (ΔglgC/xylAB), in which glycogen synthesis is blocked, and xylose catabolism enabled through the introduction of xylose isomerase and xylulokinase. We show that this mutant strain is able to metabolise xylose to acetate on nitrogen starvation. To see whether acetate production in the mutant is linked to the activity of phosphoketolase, we disrupted a putative phosphoketolase gene (slr0453) in the ΔglgC/xylAB strain, and monitored metabolic flux using (13)C labelling; acetate and 2-oxoglutarate production was reduced in the light. A metabolic flux analysis, based on isotopic data, suggests that the phosphoketolase pathway metabolises over 30% of the carbon consumed by ΔglgC/xylAB during photomixotrophic growth on xylose and CO2. Disruption of the putative phosphoketolase gene in wild-type Synechocystis also led to a deficiency in acetate production in the dark, indicative of a contribution of the phosphoketolase pathway to heterotrophic metabolism. We suggest that the phosphoketolase pathway, previously uncharacterized in photosynthetic organisms, confers flexibility in energy and carbon metabolism in cyanobacteria, and could be exploited to increase the efficiency of cyanobacterial carbon metabolism and photosynthetic productivity.
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Aldeído Liases/metabolismo , Carbono/metabolismo , Synechocystis/metabolismo , Acetatos/metabolismo , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Teste de Complementação Genética , Processos Heterotróficos , Ácidos Cetoglutáricos/metabolismo , Nitrogênio/metabolismo , Pentosefosfatos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Synechocystis/genética , Xilose/metabolismoRESUMO
BACKGROUND: Lignocellulosic biomass is one of the most promising renewable and clean energy resources to reduce greenhouse gas emissions and dependence on fossil fuels. However, the resistance to accessibility of sugars embedded in plant cell walls (so-called recalcitrance) is a major barrier to economically viable cellulosic ethanol production. A recent report from the US National Academy of Sciences indicated that, "absent technological breakthroughs", it was unlikely that the US would meet the congressionally mandated renewable fuel standard of 35 billion gallons of ethanol-equivalent biofuels plus 1 billion gallons of biodiesel by 2022. We here describe the properties of switchgrass (Panicum virgatum) biomass that has been genetically engineered to increase the cellulosic ethanol yield by more than 2-fold. RESULTS: We have increased the cellulosic ethanol yield from switchgrass by 2.6-fold through overexpression of the transcription factor PvMYB4. This strategy reduces carbon deposition into lignin and phenolic fermentation inhibitors while maintaining the availability of potentially fermentable soluble sugars and pectic polysaccharides. Detailed biomass characterization analyses revealed that the levels and nature of phenolic acids embedded in the cell-wall, the lignin content and polymer size, lignin internal linkage levels, linkages between lignin and xylans/pectins, and levels of wall-bound fucose are all altered in PvMYB4-OX lines. Genetically engineered PvMYB4-OX switchgrass therefore provides a novel system for further understanding cell wall recalcitrance. CONCLUSIONS: Our results have demonstrated that overexpression of PvMYB4, a general transcriptional repressor of the phenylpropanoid/lignin biosynthesis pathway, can lead to very high yield ethanol production through dramatic reduction of recalcitrance. MYB4-OX switchgrass is an excellent model system for understanding recalcitrance, and provides new germplasm for developing switchgrass cultivars as biomass feedstocks for biofuel production.
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Large populations of potential cellulosic biomass feedstocks are currently being screened for fuel and chemical applications. The monomeric sugar content, released through hydrolysis, is of particular importance and is currently measured with time-consuming HPLC methods. A method for sugar detection is presented here that employs (1)H NMR spectra regressed against primary HPLC sugar concentration data to build partial least squares (PLS) models. The PLS2 model is able to predict concentrations of both major sugar components, like glucose and xylose, and minor sugars, such as arabinose and mannose, in biomass hydrolysates. The model was built with 65 samples from a variety of different biomass species and covers a wide range of sugar concentrations. Model predictions were validated with a set of 15 samples which were all within error of both HPLC and NMR integration measurements. The data collection time for these NMR measurements is less than 20 min, offering a significant improvement to the 1 h acquisition time that is required for HPLC.
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Biomassa , Carboidratos/análise , Celulose/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Cromatografia Líquida de Alta Pressão/métodos , Hidrólise , Fatores de TempoRESUMO
The atomic specificity afforded by nuclear magnetic resonance (NMR) spectroscopy could enable detailed mechanistic information about single-walled carbon nanotube (SWCNT) functionalization as well as the noncovalent molecular interactions that dictate ground-state charge transfer and separation by electronic structure and diameter. However, to date, the polydispersity present in as-synthesized SWCNT populations has obscured the dependence of the SWCNT (13)C chemical shift on intrinsic parameters such as diameter and electronic structure, meaning that no information is gleaned for specific SWCNTs with unique chiral indices. In this article, we utilize a combination of (13)C labeling and density gradient ultracentrifugation (DGU) to produce an array of (13)C-labeled SWCNT populations with varying diameter, electronic structure, and chiral angle. We find that the SWCNT isotropic (13)C chemical shift decreases systematically with increasing diameter for semiconducting SWCNTs, in agreement with recent theoretical predictions that have heretofore gone unaddressed. Furthermore, we find that the (13)C chemical shifts for small diameter metallic and semiconducting SWCNTs differ significantly, and that the full-width of the isotropic peak for metallic SWCNTs is much larger than that of semiconducting nanotubes, irrespective of diameter.
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Espectroscopia de Ressonância Magnética/métodos , Nanotubos de Carbono , Isótopos de Carbono , Espectrofotometria Ultravioleta , Análise Espectral Raman , UltracentrifugaçãoRESUMO
The average spatial dimensions between major biopolymers within the plant cell wall can be resolved using a solid-state NMR technique referred to as a (13)C cross-polarization (CP) SELDOM (selectively by destruction of magnetization) with a mixing time delay for spin diffusion. Selective excitation of specific aromatic lignin carbons indicates that lignin is in close proximity to hemicellulose followed by amorphous and finally crystalline cellulose. (13)C spin diffusion time constants (T(SD)) were extracted using a two-site spin diffusion theory developed for (13)C nuclei under magic angle spinning (MAS) conditions. These time constants were then used to calculate an average lower-limit spin diffusion length between chemical groups within the plant cell wall. The results on untreated (13)C enriched corn stover stem reveal that the lignin carbons are, on average, located at distances â¼0.7-2.0 nm from the carbons in hemicellulose and cellulose, whereas the pretreated material had larger separations.