Research and development activities directed toward commercial production of cellulosic ethanol have created the opportunity to dramatically increase the transformation of lignin to value-added products. Here, we highlight recent advances in this lignin valorization effort. Discovery of genetic variants in native populations of bioenergy crops and direct manipulation of biosynthesis pathways have produced lignin feedstocks with favorable properties for recovery and downstream conversion. Advances in analytical chemistry and computational modeling detail the structure of the modified lignin and direct bioengineering strategies for future targeted properties. Refinement of biomass pretreatment technologies has further facilitated lignin recovery, and this coupled with genetic engineering will enable new uses for this biopolymer, including low-cost carbon fibers, engineered plastics and thermoplastic elastomers, polymeric foams, fungible fuels, and commodity chemicals.
The primary obstacle to producing renewable fuels from lignocellulosic biomass is a plant's recalcitrance to releasing sugars bound in the cell wall. From a sample set of wood cores representing 1,100 individual undomesticated Populus trichocarpa trees, 47 extreme phenotypes were selected across measured lignin content and ratio of syringyl and guaiacyl units (S/G ratio). This subset was tested for total sugar release through enzymatic hydrolysis alone as well as through combined hot-water pretreatment and enzymatic hydrolysis using a high-throughput screening method. The total amount of glucan and xylan released varied widely among samples, with total sugar yields of up to 92% of the theoretical maximum. A strong negative correlation between sugar release and lignin content was only found for pretreated samples with an S/G ratio < 2.0. For higher S/G ratios, sugar release was generally higher, and the negative influence of lignin was less pronounced. When examined separately, only glucose release was correlated with lignin content and S/G ratio in this manner, whereas xylose release depended on the S/G ratio alone. For enzymatic hydrolysis without pretreatment, sugar release increased significantly with decreasing lignin content below 20%, irrespective of the S/G ratio. Furthermore, certain samples featuring average lignin content and S/G ratios exhibited exceptional sugar release. These facts suggest that factors beyond lignin and S/G ratio influence recalcitrance to sugar release and point to a critical need for deeper understanding of cell-wall structure before plants can be rationally engineered for reduced recalcitrance and efficient biofuels production. L ignocellulosic biomass is the only sustainable resource in terms of cost, availability, and scale that can be converted into liquid fuels to reduce the prevailing role of petroleum in providing energy for the world's transportation needs (1, 2) and to decrease the emissions of fossil CO 2 that damage the world's climate (3). The primary obstacle to producing liquid transportation fuels by bioconversion methods is the release of sugars in high quantities at low costs from recalcitrant lignocellulosic biomass feedstocks (4, 5). Genetic modification of plants to make them less recalcitrant is a promising path to address this challenge on the feedstock side, but the effort would be greatly aided by improving understanding of the fundamental relationship between cell-wall composition and sugar release through pretreatment and enzymatic hydrolysis.In this paper, we focus on the influence of lignin content and the ratio of its syringyl and guaiacyl units (S/G ratio) on recalcitrance to sugar release, because these two traits were previously identified as dominant factors (6). Although it is generally perceived that low lignin contents increase the ability of cellulolytic enzymes to hydrolyze plant fibers (7-11), only a limited number of studies investigated the effect of lignin S/G ratio on sugar release through combined pretreatment and enzymatic hydrolysis. A...
Attempts to correlate the physical and chemical properties of biomass to its susceptibility to enzyme digestion are often inconclusive or contradictory depending on variables such as the type of substrate, the pretreatment conditions and measurement techniques. In this study, we present a direct method for measuring the key factors governing cellulose digestibility in a biomass sample by directly probing cellulase binding and activity using a purified cellobiohydrolase (Cel7A) from Trichoderma reesei. Fluorescence-labeled T. reesei Cel7A was used to assay pretreated corn stover samples and pure cellulosic substrates to identify barriers to accessibility by this important component of cellulase preparations. The results showed cellulose conversion improved when T. reesei Cel7A bound in higher concentrations, indicating that the enzyme had greater access to the substrate. Factors such as the pretreatment severity, drying after pretreatment, and cellulose crystallinity were found to directly impact enzyme accessibility. This study provides direct evidence to support the notion that the best pretreatment schemes for rendering biomass more digestible to cellobiohydrolase enzymes are those that improve access to the cellulose in biomass cell walls, as well as those able to reduce the crystallinity of cell wall cellulose.
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.
Very few cultivated microorganisms can degrade lignocellulosic biomass without chemical pretreatment. We show here that "Anaerocellum thermophilum" DSM 6725, an anaerobic bacterium that grows optimally at 75°C, efficiently utilizes various types of untreated plant biomass, as well as crystalline cellulose and xylan. These include hardwoods such as poplar, low-lignin grasses such as napier and Bermuda grasses, and high-lignin grasses such as switchgrass. The organism did not utilize only the soluble fraction of the untreated biomass, since insoluble plant biomass (as well as cellulose and xylan) obtained after washing at 75°C for 18 h also served as a growth substrate. The predominant end products from all growth substrates were hydrogen, acetate, and lactate. Glucose and cellobiose (on crystalline cellulose) and xylose and xylobiose (on xylan) also accumulated in the growth media during growth on the defined substrates but not during growth on the plant biomass. A. thermophilum DSM 6725 grew well on first-and second-spent biomass derived from poplar and switchgrass, where spent biomass is defined as the insoluble growth substrate recovered after the organism has reached late stationary phase. No evidence was found for the direct attachment of A. thermophilum DSM 6725 to the plant biomass. This organism differs from the closely related strain A. thermophilum Z-1320 in its ability to grow on xylose and pectin. Caldicellulosiruptor saccharolyticus DSM 8903 (optimum growth temperature, 70°C), a close relative of A. thermophilum DSM 6725, grew well on switchgrass but not on poplar, indicating a significant difference in the biomass-degrading abilities of these two otherwise very similar organisms.
BackgroundThe 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.ResultsGAUT12/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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0218-y) contains supplementary material, which is available to authorized users.
Summary• An association genetics approach was used to examine individual genes and alleles at the loci responsible for complex traits controlling lignocellulosic biosynthesis in black cottonwood (Populus trichocarpa). Recent interest in poplars as a source of renewable energy, combined with the vast genomic resources available, has enabled further examination of their genetic diversity.• Forty candidate genes were resequenced in a panel of 15 unrelated individuals to identify single nucleotide polymorphisms (SNPs). Eight hundred and seventy-six SNPs were successfully genotyped in a clonally replicated population (448 clones). The association population (average of 2.4 ramets per clone) was phenotyped using pyrolysis molecular beam mass spectrometry. Both single-marker and haplotype-based association tests were implemented to identify associations for composite traits representing lignin content, syringyl : guaiacyl ratio and C6 sugars.• Twenty-seven highly significant, unique, single-marker associations (false discovery rate Q < 0.10) were identified across 40 candidate genes in three composite traits. Twenty-three significant haplotypes within 11 genes were discovered in two composite traits.• Given the rapid decay of within-gene linkage disequilibrium and the high coverage of amplicons across each gene, it is likely that the numerous polymorphisms identified are in close proximity to the causative SNPs and the haplotype associations reflect information present in the associations between markers.
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