Improved Saccharification Efficiency Research Articles

Modifying lignin composition and xylan O-acetylation induces changes in cell wall composition, extractability, and digestibility

BackgroundLignin and xylan are important determinants of cell wall structure and lignocellulosic biomass digestibility. Genetic manipulations that individually modify either lignin or xylan structure improve polysaccharide digestibility. However, the effects of their simultaneous modifications have not been explored in a similar context. Here, both individual and combinatorial modification in xylan and lignin was studied by analysing the effect on plant cell wall properties, biotic stress responses and integrity sensing.ResultsArabidopsis plant co-harbouring mutation in FERULATE 5-HYDROXYLASE (F5H) and overexpressing Aspergillus niger acetyl xylan esterase (35S:AnAXE1) were generated and displayed normal growth attributes with intact xylem architecture. This fah1-2/35S:AnAXE1 cross was named as hyper G lignin and hypoacetylated (HrGHypAc) line. The HrGHypAc plants showed increased crystalline cellulose content with enhanced digestibility after chemical and enzymatic pre-treatment. Moreover, both parents and HrGHypAc without and after pre-treating with glucuronyl esterase and alpha glucuronidase exhibited an increase in xylose release after xylanase digestion as compared to wild type. The de-pectinated fraction in HrGHypAc displayed elevated levels of xylan and cellulose. Furthermore, the transcriptomic analysis revealed differential expression in cell wall biosynthetic, transcription factors and wall-associated kinases genes implying the role of lignin and xylan modification on cellular regulatory processes.ConclusionsSimultaneous modification in xylan and lignin enhances cellulose content with improved saccharification efficiency. These modifications loosen cell wall complexity and hence resulted in enhanced xylose and xylobiose release with or without pretreatment after xylanase digestion in both parent and HrGHypAc. This study also revealed that the disruption of xylan and lignin structure is possible without compromising either growth and development or defense responses against Pseudomonas syringae infection.

Pullulanase pretreatment of highly concentrated maltodextrin solution improves maltose yield during β-amylase-catalyzed saccharification

Increasing the substrate concentration can effectively reduce energy consumption and result in more economic benefits in the industrial production of maltose, but this process remarkably increases the viscosity, which has a negative effect on saccharification. To improve saccharification efficiency, pullulanase is usually employed. In the conventional process of maltose production, pullulanase is added at the same time with β-amylase or later, but this process seems inefficient when the substrate concentration is high. Herein, a novel method was introduced to enhance the maltose yield under high substrate concentration. The results indicated that the pullulanase pretreatment of highly concentrated maltodextrin solution for 2 h greatly affects the final conversion rate of β-amylase-catalyzed saccharification. The maltose yield reached 80.95 %, which is 11.8 % above the control value. Further examination confirmed that pullulanase pretreatment decreased the number of branch points of maltodextrin and resulted in a high content of oligosaccharides. These linear chains were suitable for β-amylase-catalyzed saccharification to produce maltose. This research offers a new effective and green strategy for starch sugar production.

A comprehensive review on genetic modification of plant cell wall for improved saccharification efficiency.

The focus is now on harnessing energy from green sources through sustainable technology to minimize environmental pollution. Several crop residues including rice and wheat straw are having enormous potential to be used as lignocellulosic source material for bioenergy production. The lignocellulosic feedstock is primarily composed of cellulose, hemicellulose, and lignin cell wall polymers. The hemicellulose and lignin polymers induce crosslinks in the cell wall, by firmly associating with cellulose microfibrils, and thereby, denying considerable access of cellulose to cellulase enzymes. This issue has been addressed by various researchers through downregulating several genes associated in monolignol biosynthesis in Arabidopsis, Poplar, Rice and Switchgrass to increase ethanol recovery. Similarly, xylan biosynthetic genes are also targeted to genetically culminate its accumulation in the secondary cell walls. Regulation of cellulose synthases (CesA) proves to be an effective tool in addressing the negative impact of these two factors. Modification in the expression of cellulose synthase aids in reducing cellulose crystallinity as well as polymerisation degree which in turn increases ethanol recovery. The engineered bioenergy crops and various fungal strains with state of art biomass processing techniques presents the most recent integrative biotechnology model for cost effective green fuels generation along with production of key value-added products with minuscule disturbances in the environment. Plant breeding strategies utilizing the existing variability for biomass traits will be key in developing dual purpose varieties. For this purpose, reorientation of conventional breeding techniques for incorporating useful biomass traits will be effective.

Improvement of Large-Scale Production of Lignocellulosic Bioethanol through Synthetic Biology Approaches: A Comprehensive Review

This review article explores the use of synthetic biology approaches like gene editing and metabolic engineering to create genetically-modified organisms that can produce commercially-valuable products on large-scale, clean environmental pollutants, and serve as efficient biosensors. The Biobrick system includes a registry of available genetic information and a set of standard protocols for assembling and testing them, which finds various significant uses in synthetic biology, such as in the production of "Fourth generation” biofuel. Genetic engineering techniques may be applied to manipulate crops or microbes for more efficient biofuel production. Such synthetic biology techniques also include modification of lignin structure and content, use of bacterial enzymes abundantly expressed in plants, and manipulation of hemicellulose biosynthesis. However, challenges such as high temperature, efficient conversion of all substrate molecules for maximum product yield, efficient pentose fermentation, and tolerance to acetic acid and bioethanol concentration can all impact the yield of the product. This article explores innovative genetic modification approaches designed to decrease lignin levels and enhance crop digestibility in plants, with a specific focus on the model plant Arabidopsis thaliana and herbaceous plants such as Alfalfa. The aim is to manipulate the Monolignol pathway and modify the CSE gene, known for its ability to decrease lignin content. These modifications have been shown to significantly improve saccharification efficiency and increase the yield of lignocellulosic bioethanol. Besides, the role of xylulose-5-phosphate as an intermediate in the non-oxidative pentose phosphate pathway and the oxidative pentose phosphate pathway, and the use of heterologous xylose transporters and modified sugar transporters as potential solutions to improve the biofuel production efficiency is also discussed in this review article.

Assessment of pure, mixed and diluted deep eutectic solvents on Napier grass (Cenchrus purpureus): Compositional and characterization studies of cellulose, hemicellulose and lignin

Pretreatment with pure, mixed, and diluted deep eutectic solvents (DESs) was evaluated for its effect on Napier grass through compositional and characterization studies. The morphological changes of biomass caused by pretreatment were analyzed by FTIR and XRD. The cellulose and hemicellulose content after pretreatment using mixed DES increased and decreased 1.29- and 4.25-fold, respectively, when compared to untreated Napier grass. The crystallinity index (CrI. %) of mixed DES sample increased due to the maximum removal of hemicellulose (76 %) and delignification of 62 %. The material costs of ChCl/FA and ChCl/LA for a single run are ≈2.16 USD and ≈1.65 USD, respectively. Pure DES showed that ChCl/LA pretreatment enhanced delignification efficiency and that ChCl/FA increased hemicellulose removal. It was estimated that a single run using ChCl/LA:ChCl/FA to achieve maximum hemicellulose and lignin removal would cost approximately ≈1.89 USD. Future work will evaluate the effect of DES mixture on enzyme digestibility and ethanol production from Napier grass. HypothesesDeep eutectic solvent (DES) pretreatment studies on the fractionation of lignocellulosic biomass have grown exponentially. The use of pure and diluted DES has been reported to improve saccharification efficiency, delignification, and cellulose retention (Gundupalli et al., 2022). These studies have reported maximum lignin removal but also a lower effect on hemicellulose removal from lignocellulosic biomass. It was hypothesized that mixing two pure DESs could result in maximum removal of hemicellulose and lignin after pretreatment. To our knowledge, no studies have been performed to investigate the efficiency of pretreatment using a DES mixture and compared the outcome with pure and diluted DESs. Furthermore, it was hypothesized that using two pure DESs in a mixed form could lower the material cost for each experimental run. Process efficiency was determined by compositional, XRD, and FTIR analysis. Avenues for future research include determining glucose and ethanol yields during the enzymatic saccharification and fermentation processes.

Optimization of alkali pretreatment and enzymatic saccharification of jute (Corchorus olitorius L.) biomass using response surface methodology

Reduction of inherent structural recalcitrance and improved saccharification efficiency are two important facets to enhance fermentable sugar yield for bioethanol production from lignocellulosic biomass. This study optimized alkaline pretreatment and saccharification conditions employing response surface methodology to improve saccharification yield of jute (Corchorus olitorius cv. JROB-2) biomass. The biomass is composed of cellulose (66.6 %), lignin (19.4 %) and hemicellulose (13.1 %). NaOH concentration exhibited significant effect on delignification during pretreatment. The highest delignification (80.42 %) was obtained by pretreatment with 2.47 % NaOH at 55.8 °C for 5.9 h removing 79.8 % lignin and 34.2 % hemicellulose from biomass, thereby increasing cell wall porosity and allowing better accessibility to saccharification enzyme. During saccharification optimization, significant effect was observed for biomass loading, enzyme concentration and temperature. Optimized saccharification condition yielded maximum saccharification (76.48 %) when hydrolysis was performed at 6.9 % biomass loading with enzyme concentration of 49.52 FPU/g substrate at 51.05 °C for 74.46 h.

Manipulation of Lignin Monomer Composition Combined with the Introduction of Monolignol Conjugate Biosynthesis Leads to Synergistic Changes in Lignin Structure

The complexity of lignin structure impedes efficient cell wall digestibility. Native lignin is composed of a mixture of three dominant monomers, coupled together through a variety of linkages. Work over the past few decades has demonstrated that lignin composition can be altered through a variety of mutational and transgenic approaches such that the polymer is derived almost entirely from a single monomer. In this study, we investigated changes to lignin structure and digestibility in Arabidopsis thaliana in near-single-monolignol transgenics and mutants and determined whether novel monolignol conjugates, produced by a FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) or a p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (PMT), could be integrated into these novel polymers to further improve saccharification efficiency. Monolignol conjugates, including a new conjugate of interest, p-coumaryl p-coumarate, were successfully integrated into high-H, high-G and high-S lignins in A. thaliana. Regardless of lignin composition, FMT- and PMT-expressing plants produced monolignol ferulates and monolignol p-coumarates, respectively, and incorporated them into their lignin. Through the production and incorporation of monolignol conjugates into near-single-monolignol lignins, we demonstrated that substrate availability, rather than monolignol transferase substrate preference, is the most important determining factor in the production of monolignol conjugates, and lignin composition helps dictate cell wall digestibility.

Combined effect of hot water and deep eutectic solvent (DES) pretreatment on a lignocellulosic biomass mixture for improved saccharification efficiency

The combined effect of hot water (HW) and deep eutectic solvent (DES) on the solubilization of hemicellulose and lignin present in a lignocellulose mixture (rice straw, napier grass, and sugarcane bagasse in a 1:1:1 ratio) was investigated. The selected DES for present study included choline chloride (ChCl) with urea (U), glycerol (G), and lactic acid (LA) at a molar ratio of 1:2 w/w. The selection of different lignocellulosic biomass was made taking into account the similar percentage of hemicellulose and lignin. The hot water and DES pretreatment was performed with biomass loading of 1:10 w/v, 121 °C, 40 min. and 130 °C, 95 min, and 150 rpm, respectively. The HW-ChCl-LA resulted in 4.51-fold increase in cellulose digestibility at 72 h (20.63 ± 0.68% to 93 ± 3.62%). The FTIR spectrum for pretreated solids showed significant changes in the wavelength related to hemicellulose and lignin.

Polystyrene sulfonate is effective for enhancing biomass enzymatic saccharification under green liquor pretreatment in bioenergy poplar

BackgroundWater-soluble lignin (particularly lignosulfonate, LS) has been well documented for its significance on enzymatic saccharification of lignocellulose, though the promotion mechanism has not been fully understood. Much attention has been paid to natural lignin or its derivatives. The disadvantage of using natural lignin-based polymers as promoting agents lies in the difficulty in tailor-incorporating functional groups due to their complex 3D structures. To further improve our understanding on the promotion mechanism of water-soluble lignin in the bioconversion of lignocellulose and to pursue better alternatives with different skeleton structures other than natural lignin, herein we reported a synthetic soluble linear aromatic polymer, sodium polystyrene sulfonate (PSS), to mimic LS for enhancing the efficiency of enzymatic saccharification.ResultsThe role of PSS in enzymatic saccharification of pure cellulose and green liquor-pretreated poplar (GL-P) was explored by analyzing substrate enzymatic digestibility (SED) under different addition dosages and various pH media, along with LS for comparison. At the cellulase loading of 13.3 FPU/g-glucan, the glucose yield of GL-P increased from 53% for the control to 81.5% with PSS addition of 0.1 g/g-substrate. It outperformed LS with the addition of 0.2 g/g-substrate by 6.3%. In the pH range from 4.5 to 6, PSS showed a positive effect on lignocellulose saccharification with the optimum pH at 4.8, where the most pronounced SED of GL-P was achieved. The underlying mechanism was unveiled by measuring zeta potential and using Quartz Crystal Microbalance (QCM) and Multi-parametric Surface Plasmon Resonance (MP-SPR). The results confirmed that the complexes of cellulase and PSS were conjugated and the negatively supercharged complexes reduced non-productive binding effectively along with the improved saccharification efficiency. The thickness of PSS required to block the binding sites of cellulase film was less than half of that of LS, and the PSS adlayer on cellulase film is also more hydrated and with a much lower shear modulus than LS adlayer.ConclusionsPSS as LS analogue is effective for enhancing the biomass enzymatic saccharification of GL-pretreated poplar. PSS exhibited a severer inhibition on the enzymatic saccharification of pure cellulose, while a more positive effect on bioconversion of lignocellulose (GL-P) than LS. In addition, a much lower dosage is required by PSS. The dynamic enzymatic hydrolysis indicated PSS could prolong the processive activity of cellulase. The valid data stemmed from QCM and SPR expressed that PSS bound to cellulases and the as-formed complexes reduced the non-productive adsorption of cellulase onto substrate lignin more efficiently than LS due to its flexible skeleton and highly hydrated structure. Therefore, PSS is a promising alternative promoting agent for lignocellulose saccharification. From another perspective, the synthetic lignin mimics with controllable structures enable us to reach an in-depth understanding of the promotion mechanism of soluble lignins on enzymatic saccharification.

Field evaluation of transgenic hybrid poplars with desirable wood properties and enhanced growth for biofuel production by bicistronic expression of PdGA20ox1 and PtrMYB3 in wood-forming tissue

BackgroundTo create an ideotype woody bioenergy crop with desirable growth and biomass properties, we utilized the viral 2A-meidated bicistronic expression strategy to express both PtrMYB3 (MYB46 ortholog of Populus trichocarpa, a master regulator of secondary wall biosynthesis) and PdGA20ox1 (a GA20-oxidase from Pinus densiflora that produces gibberellins) in wood-forming tissue (i.e., developing xylem).ResultsTransgenic Arabidopsis plants expressing the gene construct DX15::PdGA20ox1-2A-PtrMYB3 showed a significant increase in both stem fresh weight (threefold) and secondary wall thickening (1.27-fold) relative to wild-type (WT) plants. Transgenic poplars harboring the same gene construct grown in a greenhouse for 60 days had a stem fresh weight up to 2.6-fold greater than that of WT plants. In a living modified organism (LMO) field test conducted for 3 months of active growing season, the stem height and diameter growth of the transgenic poplars were 1.7- and 1.6-fold higher than those of WT plants, respectively, with minimal adverse growth defects. Although no significant changes in secondary wall thickening of the stem tissue of the transgenic poplars were observed, cellulose content was increased up to 14.4 wt% compared to WT, resulting in improved saccharification efficiency of the transgenic poplars. Moreover, enhanced woody biomass production by the transgenic poplars was further validated by re-planting in the same LMO field for additional two growing seasons.ConclusionsTaken together, these results show considerably enhanced wood formation of our transgenic poplars, with improved wood quality for biofuel production.

Bioconversion of renewable lignocellulosic biomass into multicomponent substrate via pressurized hot water pretreatment for bioplastic polyhydroxyalkanoate accumulation

The pretreatment of lignocellulosic biomass (LB) has become an important process to reduce the cost of polyhydroxyalkanoate (PHA) production. In this study, an economical and effective pressurized hot water pretreatment was used to investigate on bioconversion of four typical LB (rubber wood, sugarcane bagasse, sorghum stalk, cassava stalk) into reducing sugar, then as feedstock to accumulate PHA by mixed microbial cultures. The results showed that pretreatment changed the surface morphology of LB and further improved saccharification efficiency. The maximum reducing sugar yield of 620.7 mg·g−1 (438.7 mg·g−1 glucose and 178.0 mg·g−1 xylose) was obtained by rubber wood. The conversion of cellulose and hemicellulose reached 83.10% and 78.22%. Moreover, PHA content reached the maximum of 773.2 mg COD·L−1 in the operation cycle of 24 h. The results demonstrated that hot water pretreatment was an effective physical process to improve the saccharification efficiency of LB for reducing the cost of PHA.

Improving enzymatic digestibility of sugarcane bagasse from different varieties of sugarcane using deep eutectic solvent pretreatment

Sugarcane bagasse, a fundamental by-product of the sugar industry, was utilised to improve its digestibility for bioenergy applications. Choline chloride based deep eutectic solvents (DESs) were used for pretreatment of five different varieties of sugarcane bagasse (SRA1, SRA5, Q208, MA239, ISB) and a comparative study of compositional and morphological changes was performed. Three eutectic mixtures - choline chloride: malonic acid (1:1), choline chloride: glycerol (1:2) and choline chloride: lactic acid (1:5) were used to selectively remove lignin and improve saccharification efficiency. Physico-chemical characterizations performed using FE-SEM, FTIR, TGA and XRD analysis consistently indicated disruption of bagasse structure after DES pretreatment. Glucose recovery was predominantly influenced by the glucose content, as SRA1 variety showed the highest recovery of 92.8% for choline chloride: glycerol DES pretreatment. Choline chloride: lactic acid DES pretreatment demonstrated the most efficient lignin removal of 81.6% for ISB variety and the enzyme amenability was prominently increased to 98.5%.

MicroRNA408 Enhances Growth and Saccharification Efficiency Through Altering the Balance between Vascular Development and Lignification in Hybrid Poplar

Saccharification, the conversion of cellulose to sugar for fermentation-based biofuels, remains problematic for lignocellulosic feedstocks such as wood. Genetic modifications to reduce the amount or change the structure of lignin can improve saccharification efficiency but often result in dwarf plants. Here, we report that overexpression of Pag-MIR408 in hybrid poplar results in a near doubling saccharification efficiency, with no requirement for acid-pretreatment, increased accessibility of secondary cell walls to fungal cellulase and scaffoldin cellulose-binding modules, accompanied by enhanced photosynthesis, and increased number of cambium zone layers, xylem width and vessel diameter. Conversely, Pag-MIR408 loss-of-function plants by CRISPR/Cas9 showed decreased cell wall accessibility. Moreover, miR408 targets LACCASE 19/25, leading to delayed lignification, and modestly reduced lignin content, S/G ratio, as well as the degree of lignin polymerization. This study provides a unique insight into enhanced saccharification efficiency altered wood structure through modulation of the balance between secondary development and lignification.

Association mapping for maize stover yield and saccharification efficiency using a multiparent advanced generation intercross (MAGIC) population

Cellulosic ethanol derived from fast growing C4 grasses could become an alternative to finite fossil fuels. With the potential to generate a major source of lignocellulosic biomass, maize has gained importance as an outstanding model plant for studying the complex cell wall network and also to optimize crop breeding strategies in bioenergy grasses. A genome-wide association study (GWAS) was conducted using a subset of 408 Recombinant Inbred Lines (RILs) from a Multi-Parent Advanced Generation Intercross (MAGIC) Population in order to identify single nucleotide polymorphisms (SNPs) associated with yield and saccharification efficiency of maize stover. We identified 13 SNPs significantly associated with increased stover yield that corresponded to 13 QTL, and 2 SNPs significantly associated with improved saccharification efficiency, that could be clustered into 2 QTL. We have pointed out the most interesting SNPs to be implemented in breeding programs based on results from analyses of averaged and yearly data. Association mapping in this MAGIC population highlight genomic regions directly linked to traits that influence the final use of maize. Markers linked to these QTL could be used in genomic or marker-assisted selection programs to improve biomass quality for ethanol production. This study opens a possible optimisation path for improving the viability of second-generation biofuels.

The known unknowns in lignin biosynthesis and its engineering to improve lignocellulosic saccharification efficiency

Biofuels produced from lignocellulosic biomass provide energy security and reduce greenhouse gas emissions with positive impacts on sustainability. Lignocellulose byproducts are expected to impact the production of cellulosic ethanol. However, lignin is a major factor imposing biomass recalcitrance to saccharification. Although structural phenolics can be removed from lignocellulosic biomass, physical and chemical pretreatments are expensive. In turn, the structure and composition of lignin is very flexible. Up- and downregulation of genes and, thereby, enzymes involved in core and non-core lignin biosynthesis can reduce lignin content and improve biomass saccharification. Alternatively, insertion of heterologous genes to the phenylpropanoid pathway may lead to the incorporation of alternative monomers into lignin, creating desirable properties and preserving the biological roles of lignin with a positive effect on saccharification. Studies reporting the effect of fine regulation on lignin structures are mapping how plants can be transformed to enhance saccharification while preserving productivity. Based on these findings and events, this review updates the state-of-art changes in lignin biosynthesis to improve saccharification efficiency. The main findings reported over the last decade were summarized to permit researchers an overview of this relevant scientific subject.

Rewired phenolic metabolism and improved saccharification efficiency of a Zea mays cinnamyl alcohol dehydrogenase 2 (zmcad2) mutant.

Lignocellulosic biomass is an abundant byproduct from cereal crops that can potentially be valorized as a feedstock to produce biomaterials. Zea mays CINNAMYL ALCOHOL DEHYDROGENASE 2 (ZmCAD2) is involved in lignification, and is a promising target to improve the cellulose-to-glucose conversion of maize stover. Here, we analyzed a field-grown zmcad2 Mutator transposon insertional mutant. Zmcad2 mutant plants had an 18% lower Klason lignin content, whereas their cellulose content was similar to that of control lines. The lignin in zmcad2 mutants contained increased levels of hydroxycinnamaldehydes, i.e. the substrates of ZmCAD2, ferulic acid and tricin. Ferulates decorating hemicelluloses were not altered. Phenolic profiling further revealed that hydroxycinnamaldehydes are partly converted into (dihydro)ferulic acid and sinapic acid and their derivatives in zmcad2 mutants. Syringyl lactic acid hexoside, a metabolic sink in CAD-deficient dicot trees, appeared not to be a sink in zmcad2 maize. The enzymatic cellulose-to-glucose conversion efficiency was determined after 10 different thermochemical pre-treatments. Zmcad2 yielded significantly higher conversions compared with controls for almost every pre-treatment. However, the relative increase in glucose yields after alkaline pre-treatment was not higher than the relative increase when no pre-treatment was applied, suggesting that the positive effect of the incorporation of hydroxycinnamaldehydes was leveled off by the negative effect of reduced p-coumarate levels in the cell wall. Taken together, our results reveal how phenolic metabolism is affected in CAD-deficient maize, and further support mutating CAD genes in cereal crops as a promising strategy to improve lignocellulosic biomass for sugar-platform biorefineries.

Identifying transcription factors that reduce wood recalcitrance and improve enzymatic degradation of xylem cell wall in Populus

Developing an efficient deconstruction step of woody biomass for biorefinery has been drawing considerable attention since its xylem cell walls display highly recalcitrance nature. Here, we explored transcriptional factors (TFs) that reduce wood recalcitrance and improve saccharification efficiency in Populus species. First, 33 TF genes up-regulated during poplar wood formation were selected as potential regulators of xylem cell wall structure. The transgenic hybrid aspens (Populus tremula × Populus tremuloides) overexpressing each selected TF gene were screened for in vitro enzymatic saccharification. Of these, four transgenic seedlings overexpressing previously uncharacterized TF genes increased total glucan hydrolysis on average compared to control. The best performing lines overexpressing Pt × tERF123 and Pt × tZHD14 were further grown to form mature xylem in the greenhouse. Notably, the xylem cell walls exhibited significantly increased total xylan hydrolysis as well as initial hydrolysis rates of glucan. The increased saccharification of Pt × tERF123-overexpressing lines could reflect the improved balance of cell wall components, i.e., high cellulose and low xylan and lignin content, which could be caused by upregulation of cellulose synthase genes upon the expression of Pt × tERF123. Overall, we successfully identified Pt × tERF123 and Pt × tZHD14 as effective targets for reducing cell wall recalcitrance and improving the enzymatic degradation of woody plant biomass.

MicroRNA6443-mediated regulation of FERULATE 5-HYDROXYLASE gene alters lignin composition and enhances saccharification in Populus tomentosa.

Ferulate 5-hydroxylase (F5H) is a limiting enzyme involved in biosynthesizing sinapyl (S) monolignol in angiosperms. Genetic regulation of F5H can influence S monolignol synthesis and therefore improve saccharification efficiency and biofuel production. To date, little is known about whether F5H is post-transcriptionally regulated by endogenous microRNAs (miRNAs) in woody plants. Here, we report that a microRNA, miR6443, specifically regulates S lignin biosynthesis during stem development in Populus tomentosa. In situ hybridization showed that miR6443 is preferentially expressed in vascular tissues. We further identified that F5H2 is the direct target of miR6443. Overexpression of miR6443 decreased the transcript level of F5H2 in transgenic plants, resulting in a significant reduction in S lignin content. Conversely, reduced miR6443 expression by short tandem target mimics (STTM) elevated F5H2 transcripts, therefore increasing S lignin composition. Introduction of a miR6443-resistant form of F5H2 into miR6443-overexpression plants restored lignin ectopic composition, supporting that miR6443 specifically regulated S lignin biosynthesis by repressing F5H2 in P.tomentosa. Furthermore, saccharification assays revealed decreased hexose yields by 7.5-24.5% in miR6443-overexpression plants compared with the wild-type control, and increased hexoses yields by 13.2-14.6% in STTM6443-overexpression plants. Collectively, we demonstrate that miR6443 modulates S lignin biosynthesis by specially regulating F5H2 in P.tomentosa.

Isolation, cloning and expression analysis of cinnamyl alcohol dehydrogenase (CAD) involved in phenylpropanoid pathway of Erianthus arundinaceus, a wild relative of sugarcane.

In this study we have cloned and characterized cinnamyl alcohol dehydrogenase (CAD) involved in phenylpropanoid pathway which can be utilized for biomass modification for improved saccharification efficiency. The full length gene CAD is of 4kb containing four exons and three introns, among which the exon 1 and 2 of 88 and 116bp were conserved with sorghum and Miscanthus CADs. The coding region of CAD was identified with 1098bp open reading frame (ORF), for 365 amino acids. In the PROSITE analysis, a zinc-containing alcohol dehydrogenase signature (GHEVVGEVVEVGPEV) and an NADP-binding domain motif (GLGGLG) was identified, while the motif analysis showed unique signature sequence of "LEPYLA" at 258-264 aa which was absent in the CAD sequences of other crops. This sequence information on CAD from Erianthus a bioenergy crop might be useful for subsequent research on lignin engineering for improved biomass conversion and for unravelling the impact of lignin on cell wall mechanics.

Relationship between sugarcane culm and leaf biomass composition and saccharification efficiency

BackgroundLignocellulosic biomass is recognized as a promising renewable feedstock for the production of biofuels. However, current methods for converting biomass into fermentable sugars are considered too expensive and inefficient due to the recalcitrance of the secondary cell wall. Biomass composition can be modified to create varieties that are efficiently broken down to release cell wall sugars. This study focused on identifying the key biomass components influencing plant cell wall recalcitrance that can be targeted for selection in sugarcane, an important and abundant source of biomass.ResultsBiomass composition and the amount of glucan converted into glucose after saccharification were measured in leaf and culm tissues from seven sugarcane genotypes varying in fiber composition after no pretreatment and dilute acid, hydrothermal and ionic liquid pretreatments. In extractives-free sugarcane leaf and culm tissue, glucan, xylan, acid-insoluble lignin (AIL) and acid-soluble lignin (ASL) ranged from 20 to 32%, 15% to 21%, 14% to 20% and 2% to 4%, respectively. The ratio of syringyl (S) to guaiacyl (G) content in the lignin ranged from 1.5 to 2.2 in the culm and from 0.65 to 1.1 in the leaf. Hydrothermal and dilute acid pretreatments predominantly reduced xylan content, while the ionic liquid (IL) pretreatment targeted AIL reduction. The amount of glucan converted into glucose after 26 h of pre-saccharification was highest after IL pretreatment (42% in culm and 63.5% in leaf) compared to the other pretreatments. Additionally, glucan conversion in leaf tissues was approximately 1.5-fold of that in culm tissues. Percent glucan conversion varied between genotypes but there was no genotype that was superior to all others across the pretreatment groups. Path analysis revealed that S/G ratio, AIL and xylan had the strongest negative associations with percent glucan conversion, while ASL and glucan content had strong positive influences.ConclusionTo improve saccharification efficiency of lignocellulosic biomass, breeders should focus on reducing S/G ratio, xylan and AIL content and increasing ASL and glucan content. This will be key for the development of sugarcane varieties for bioenergy uses.