Rerouting of the lignin biosynthetic pathway by inhibition of cytosolic shikimate recycling in transgenic hybrid aspen.

Lignin is a phenolic polymer deposited in the plant cell wall, and is mainly polymerized from three canonical monomers (monolignols), i.e. p-coumaryl, coniferyl and sinapyl alcohols. After polymerization, these alcohols form different lignin substructures. In dicotyledons, monolignols are biosynthesized from phenylalanine, an aromatic amino acid. Shikimate acts at two positions in the route to the lignin building blocks. It is part of the shikimate pathway that provides the precursor for the biosynthesis of phenylalanine, and is involved in the transesterification of p-coumaroyl-CoA to p-coumaroyl shikimate, one of the key steps in the biosynthesis of coniferyl and sinapyl alcohols. The shikimate residue in p-coumaroyl shikimate is released in later steps, and the resulting shikimate becomes available again for the biosynthesis of new p-coumaroyl shikimate molecules. In this study, we inhibited cytosolic shikimate recycling in transgenic hybrid aspen by accelerated phosphorylation of shikimate in the cytosol through expression of a bacterial shikimate kinase (SK). This expression elicited an increase in p-hydroxyphenyl units of lignin and, by contrast, a decrease in guaiacyl and syringyl units. Transgenic plants with high SK activity produced a lignin content comparable to that in wild-type plants, and had an increased processability via enzymatic saccharification. Although expression of many genes was altered in the transgenic plants, elevated SK activity did not exert a significant effect on the expression of the majority of genes responsible for lignin biosynthesis. The present results indicate that cytosolic shikimate recycling is crucial to the monomeric composition of lignin rather than for lignin content.

Genetic complexity of miscanthus cell wall composition and biomass quality for biofuels.

BACKGROUND: Miscanthus sinensis is a high yielding perennial grass species with great potential as a bioenergy feedstock. One of the challenges that currently impedes commercial cellulosic biofuel production is the technical difficulty to efficiently convert lignocellulosic biomass into biofuel. The development of feedstocks with better biomass quality will improve conversion efficiency and the sustainability of the value-chain. Progress in the genetic improvement of biomass quality may be substantially expedited by the development of genetic markers associated to quality traits, which can be used in a marker-assisted selection program. RESULTS: To this end, a mapping population was developed by crossing two parents of contrasting cell wall composition. The performance of 182 F1 offspring individuals along with the parents was evaluated in a field trial with a randomized block design with three replicates. Plants were phenotyped for cell wall composition and conversion efficiency characters in the second and third growth season after establishment. A new SNP-based genetic map for M. sinensis was built using a genotyping-by-sequencing (GBS) approach, which resulted in 464 short-sequence uniparental markers that formed 16 linkage groups in the male map and 17 linkage groups in the female map. A total of 86 QTLs for a variety of biomass quality characteristics were identified, 20 of which were detected in both growth seasons. Twenty QTLs were directly associated to different conversion efficiency characters. Marker sequences were aligned to the sorghum reference genome to facilitate cross-species comparisons. Analyses revealed that for some traits previously identified QTLs in sorghum occurred in homologous regions on the same chromosome. CONCLUSION: In this work we report for the first time the genetic mapping of cell wall composition and bioconversion traits in the bioenergy crop miscanthus. These results are a first step towards the development of marker-assisted selection programs in miscanthus to improve biomass quality and facilitate its use as feedstock for biofuel production.

RNA interference suppression of lignin biosynthesis increases fermentable sugar yields for biofuel production from field-grown sugarcane.

The agronomic performance, cell wall characteristics and enzymatic saccharification efficiency of transgenic sugarcane plants with modified lignin were evaluated under replicated field conditions. Caffeic acid O-methyltransferase (COMT) was stably suppressed by RNAi in the field, resulting in transcript reduction of 80%-91%. Along with COMT suppression, total lignin content was reduced by 6%-12% in different transgenic lines. Suppression of COMT also altered lignin composition by reducing syringyl units and p-coumarate incorporation into lignin. Reduction in total lignin by 6% improved saccharification efficiency by 19%-23% with no significant difference in biomass yield, plant height, stalk diameter, tiller number, total structural carbohydrates or brix value when compared with nontransgenic tissue culture-derived or transgenic control plants. Lignin reduction of 8%-12% compromised biomass yield, but increased saccharification efficiency by 28%-32% compared with control plants. Biomass from transgenic sugarcane lines that have 6%-12% less lignin requires approximately one-third of the hydrolysis time or 3- to 4-fold less enzyme to release an equal or greater amount of fermentable sugar than nontransgenic plants. Reducing the recalcitrance of lignocellulosic biomass to saccharification by modifying lignin biosynthesis is expected to greatly benefit the economic competitiveness of sugarcane as a biofuel feedstock.

Approach to an efficient pretreatment method for rice straw by deep eutectic solvent for high saccharification efficiency.

Deep eutectic solvent comprising choline chloride (ChCl) and acetic acid (AA) was used for rice straw (RS) pretreatment. Effect of ChCl: AA molar ratio, time and temperature on lignin removal and retainment of total carbohydrate content (TCC) in pretreatment process were evaluated by central composite design (CCD) approach. The pretreatment temperature and molar ratio of AA to ChCl played a significant role in delignification. The optimized conditions for RS pretreatment were 1:3.59 (ChCl:AA molar ratio), 126 °C and 150 min. ChCl:AA pretreated RS (CApRS) gave 83.1% delignification, 679 mg/gCApRS TCC and 83.7% pretreatment efficiency. CApRS contained enriched cellulose content, 0.73 g/gCApRS as compared with 0.43 g/graw RS in raw RS. CApRS showed 31% higher crystallinity index, 17-fold higher surface area than raw RS. The morphological study of CApRS displayed porous surface. Saccharification of CApRS by commercial cellulase gave total reducing sugar of 18.8 g/L in hydrolysate with saccharification efficiency, 92.2%.

Targeting hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase for lignin modification in Brachypodium distachyon.

BACKGROUND: Hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) is a central enzyme of the so-called "esters" pathway to monolignols. As originally envisioned, HCT functions twice in this pathway, to form coumaroyl shikimate and then, in the "reverse" direction, to convert caffeoyl shikimate to caffeoyl CoA. The discovery of a caffeoyl shikimate esterase (CSE) that forms caffeic acid directly from caffeoyl shikimate calls into question the need for the reverse HCT reaction in lignin biosynthesis. Loss of function of HCT gives severe growth phenotypes in several dicot plants, but less so in some monocots, questioning whether this enzyme, and therefore the shikimate shunt, plays the same role in both monocots and dicots. The model grass Brachypodium distachyon has two HCT genes, but lacks a classical CSE gene. This study was therefore conducted to evaluate the utility of HCT as a target for lignin modification in a species with an "incomplete" shikimate shunt. RESULTS: The kinetic properties of recombinant B. distachyon HCTs were compared with those from Arabidopsis thaliana, Medicago truncatula, and Panicum virgatum (switchgrass) for both the forward and reverse reactions. Along with two M. truncatula HCTs, B. distachyon HCT2 had the least kinetically unfavorable reverse HCT reaction, and this enzyme is induced when HCT1 is down-regulated. Down regulation of B. distachyon HCT1, or co-down-regulation of HCT1 and HCT2, by RNA interference led to reduced lignin levels, with only modest changes in lignin composition and molecular weight. CONCLUSIONS: Down-regulation of HCT1, or co-down-regulation of both HCT genes, in B. distachyon results in less extensive changes in lignin content/composition and cell wall structure than observed following HCT down-regulation in dicots, with little negative impact on biomass yield. Nevertheless, HCT down-regulation leads to significant improvements in biomass saccharification efficiency, making this gene a preferred target for biotechnological improvement of grasses for bioprocessing.

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 4 kb containing four exons and three introns, among which the exon 1 and 2 of 88 and 116 bp were conserved with sorghum and Miscanthus CADs. The coding region of CAD was identified with 1098 bp 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.

Characteristics of a recombinant Lentinula edodes endoglucanase and its potential for application in silage of rape straw.

An experiment was conducted to determine the characteristics of recombinant endoglucanase and its effects on rape straw silage. The endoglucanase from Lentinula edodes (LeCel12A) was produced in Pichia pastoris and shown maximum activity at 40 °C and pH 3.0. The LeCel12A exhibited preferential hydrolysis of carboxymethylcellulose. The activity of LeCel12A could be enhanced by MnCl2 in dose-dependent manners. Trp22 was a key amino acid affecting LeCel12A activity. The LeCel12A enhanced the hydrolysis of rape straw, rice straw, wheat straw, and corn straw. Supplemental LeCel12A increased lactic acid concentration and reduced lignocellulosic content of the rape straw silage. Though an increase in the saccharification efficiency of LeCel12A-treated rape straw silage was observed when the fibrolytic enzyme loading of hydrolysis system was enough, supplemental LeCel12A did not dramatically enhance the saccharification of rape straw silage in the current study. This study demonstrates that LeCel12A may be useful for improving the utilization of rape straw silage as an additive, but its supplemental dose, cost benefit, and consequent application possibility in biofuel production require careful consideration and further investigation.

Relationship between sugarcane culm and leaf biomass composition and saccharification efficiency.

BACKGROUND: Lignocellulosic 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. RESULTS: Biomass 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. CONCLUSION: To 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.

Overexpression of OsPIL1 enhanced biomass yield and saccharification efficiency in switchgrass.

Switchgrass (Panicum virgatum L.) is a herbaceous cellulosic biofuel plant with broad adaptability. However, the intrinsic recalcitrance of biomass and limited land for switchgrass planting hinder its utilization as feedstock for biofuel ethanol production. The OsPIL1 (PHYTOCHROME INTERACTING FACTOR 3-LIKE 1) gene encodes a basic helix-loop-helix transcription factor. Its expression is induced by light, which facilitated the expression of cell wall-related genes, promoted cell elongation and resulted in longer internode in rice. Here, we introduced the OsPIL1 gene into switchgrass by Agrobacterium-mediated transformation with the aim of improving biomass yield of transgenic switchgrass plants. The transgenic plants were verified by PCR, Southern-blotting, RT-PCR and qRT-PCR tests, respectively. The transgenic plants overexpression of OsPIL1 showed increased plant height and biomass yield. Microscopy analysis showed that the length of epidermal cells of transgenic plants was longer than that of wild type. OsPIL1 overexpressed transgenic switchgrass plants also released more soluble sugar after enzymatic hydrolysis, indicating improved saccharification efficiency. The results suggest OsPIL1 can be used as a useful molecular tool in improving plant biomass and saccharification efficiency with the purpose of plant fiber biofuel ethanol production.