A COMPASS histone H3K4 trimethyltransferase pentamer transactivates drought tolerance and growth/biomass production in Populus trichocarpa.

Histone H3 lysine-4 trimethylation (H3K4me3) activating drought-responsive genes in plants for drought adaptation has long been established, but the underlying regulatory mechanisms are unknown. Here, using yeast two-hybrid, bimolecular fluorescence complementation, biochemical analyses, transient and CRISPR-mediated transgenesis in Populus trichocarpa, we unveiled in this adaptation a regulatory interplay between chromatin regulation and gene transactivation mediated by an epigenetic determinant, a PtrSDG2-1-PtrCOMPASS (complex proteins associated with Set1)-like H3K4me3 complex, PtrSDG2-1-PtrWDR5a-1-PtrRbBP5-1-PtrAsh2-2 (PtrSWRA). Under drought conditions, a transcription factor PtrAREB1-2 interacts with PtrSWRA, forming a PtrSWRA-PtrAREB1-2 pentamer, to recruit PtrSWRA to specific promoter elements of drought-tolerant genes, such as PtrHox2, PtrHox46, and PtrHox52, for depositing H3K4me3 to promote and maintain activated state of such genes for tolerance. CRISPR-edited defects in the pentamer impaired drought tolerance and elevated expression of PtrHox2, PtrHox46, or PtrHox52 improved the tolerance as well as growth in P. trichocarpa. Our findings revealed the identity of the underlying H3K4 trimethyltransferase and its interactive arrangement with the COMPASS for catalysis specificity and efficiency. Furthermore, our study uncovered how the H3K4 trimethyltransferase-COMPASS complex is recruited to the effector genes for elevating H3K4me3 marks for improved drought tolerance and growth/biomass production in plants.

Woody plant cell walls: Fundamentals and utilization.

Cell walls in plants, particularly forest trees, are the major carbon sink of the terrestrial ecosystem. Chemical and biosynthetic features of plant cell walls were revealed early on, focusing mostly on herbaceous model species. Recent developments in genomics, transcriptomics, epigenomics, transgenesis, and associated analytical techniques are enabling novel insights into formation of woody cell walls. Here, we review multilevel regulation of cell wall biosynthesis in forest tree species. We highlight current approaches to engineering cell walls as potential feedstock for materials and energy and survey reported field tests of such engineered transgenic trees. We outline opportunities and challenges in future research to better understand cell type biogenesis for more efficient wood cell wall modification and utilization for biomaterials or for enhanced carbon capture and storage.

Multiplex CRISPR editing of wood for sustainable fiber production.

The domestication of forest trees for a more sustainable fiber bioeconomy has long been hindered by the complexity and plasticity of lignin, a biopolymer in wood that is recalcitrant to chemical and enzymatic degradation. Here, we show that multiplex CRISPR editing enables precise woody feedstock design for combinatorial improvement of lignin composition and wood properties. By assessing every possible combination of 69,123 multigenic editing strategies for 21 lignin biosynthesis genes, we deduced seven different genome editing strategies targeting the concurrent alteration of up to six genes and produced 174 edited poplar variants. CRISPR editing increased the wood carbohydrate-to-lignin ratio up to 228% that of wild type, leading to more-efficient fiber pulping. The edited wood alleviates a major fiber-production bottleneck regardless of changes in tree growth rate and could bring unprecedented operational efficiencies, bioeconomic opportunities, and environmental benefits.

Cell-type-specific PtrWOX4a and PtrVCS2 form a regulatory nexus with a histone modification system for stem cambium development in Populus trichocarpa.

Stem vascular cambium cells in forest trees produce wood for materials and energy. WOX4 affects the proliferation of such cells in Populus. Here we show that PtrWOX4a is the most highly expressed stem vascular-cambium-specific (VCS) gene in P. trichocarpa, and its expression is controlled by the product of the second most highly expressed VCS gene, PtrVCS2, encoding a zinc finger protein. PtrVCS2 binds to the PtrWOX4a promoter as part of a PtrWOX13a-PtrVCS2-PtrGCN5-1-PtrADA2b-3 protein tetramer. PtrVCS2 prevented the interaction between PtrGCN5-1 and PtrADA2b-3, resulting in H3K9, H3K14 and H3K27 hypoacetylation at the PtrWOX4a promoter, which led to fewer cambium cell layers. These effects on cambium cell proliferation were consistent across more than 20 sets of transgenic lines overexpressing individual genes, gene-edited mutants and RNA interference lines in P. trichocarpa. We propose that the tetramer-PtrWOX4a system may coordinate genetic and epigenetic regulation to maintain normal vascular cambium development for wood formation.

Fermentative conversion of unpretreated plant biomass: A thermophilic threshold for indigenous microbial growth.

Naturally occurring, microbial contaminants were found in plant biomasses from common bioenergy crops and agricultural wastes. Unexpectedly, indigenous thermophilic microbes were abundant, raising the question of whether they impact thermophilic consolidated bioprocessing fermentations that convert biomass directly into useful bioproducts. Candidate microbial platforms for biomass conversion, Acetivibrio thermocellus (basionym Clostridium thermocellum; Topt 60 °C) and Caldicellulosiruptor bescii (Topt 78 °C), each degraded a wide variety of plant biomasses, but only A. thermocellus was significantly affected by the presence of indigenous microbial populations harbored by the biomass. Indigenous microbial growth was eliminated at ≥75 °C, conditions where C. bescii thrives, but where A. thermocellus cannot survive. Therefore, 75 °C is the thermophilic threshold to avoid sterilizing pre-treatments on the biomass that prevents native microbes from competing with engineered microbes and forming undesirable by-products. Thermophiles that naturally grow at and above 75 °C offer specific advantages as platform microorganisms for biomass conversion into fuels and chemicals.

Acute cytomegalovirus hepatitis in an immunocompetent patient: A case report.

BACKGROUND: Cytomegalovirus (CMV) infection is usually subclinical and asymptomatic in the healthy population, whereas severe complications occur in immunocompromised patients. CASE SUMMARY: In this case report, we described a rare case of acute CMV hepatitis in a 35-year-old male immunocompetent patient who presented with a history of week-long intermittent fever with nonspecific constitutional symptoms. Acute hepatitis was suspected according to the initial serological tests. After ruling out other etiologies, including viral hepatitis A, B, C, drug, alcohol, autoimmune, and Wilson disease, acute CMV hepatitis was diagnosed based on positive CMV IgM and DNA quantitative tests. Because there was no any local acute hepatitis E reported in Taiwan, so hepatitis E was not checked. The patient recovered both clinically and serologically with symptomatic management and without antiviral therapy within 12 days from the onset of symptom. CONCLUSION: In conclusion, a diagnosis of CMV infection should be considered when nonspecific prodromal symptoms occur in acute hepatitis with an uncertain etiology. Antiviral therapy should not be used in immunocompetent patient who had no decompensation of the liver, such as this patient. Widely available noninvasive tests for CMV can facilitate early diagnosis if used appropriately. Harm-benefit analysis is essential before using antiviral therapy in immunocompetent patients.

Enhancing HR Frequency for Precise Genome Editing in Plants.

Gene-editing tools, such as Zinc-fingers, TALENs, and CRISPR-Cas, have fostered a new frontier in the genetic improvement of plants across the tree of life. In eukaryotes, genome editing occurs primarily through two DNA repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is the primary mechanism in higher plants, but it is unpredictable and often results in undesired mutations, frameshift insertions, and deletions. Homology-directed repair (HDR), which proceeds through HR, is typically the preferred editing method by genetic engineers. HR-mediated gene editing can enable error-free editing by incorporating a sequence provided by a donor template. However, the low frequency of native HR in plants is a barrier to attaining efficient plant genome engineering. This review summarizes various strategies implemented to increase the frequency of HDR in plant cells. Such strategies include methods for targeting double-strand DNA breaks, optimizing donor sequences, altering plant DNA repair machinery, and environmental factors shown to influence HR frequency in plants. Through the use and further refinement of these methods, HR-based gene editing may one day be commonplace in plants, as it is in other systems.

Dimerization of PtrMYB074 and PtrWRKY19 mediates transcriptional activation of PtrbHLH186 for secondary xylem development in Populus trichocarpa.

Wood formation is controlled by transcriptional regulatory networks (TRNs) involving regulatory homeostasis determined by combinations of transcription factor (TF)-DNA and TF-TF interactions. Functions of TF-TF interactions in wood formation are still in the early stages of identification. PtrMYB074 is a woody dicot-specific TF in a TRN for wood formation in Populus trichocarpa. Here, using yeast two-hybrid and bimolecular fluorescence complementation, we conducted a genome-wide screening for PtrMYB074 interactors and identified 54 PtrMYB074-TF pairs. Of these pairs, 53 are novel. We focused on the PtrMYB074-PtrWRKY19 pair, the most highly expressed and xylem-specific interactor, and its direct transregulatory target, PtrbHLH186, the xylem-specific one of the pair's only two direct TF target genes. Using transient and CRISPR-mediated transgenesis in P. trichocarpa coupled with chromatin immunoprecipitation and electrophoretic mobility shift assays, we demonstrated that PtrMYB074 is recruited by PtrWRKY19 and that the PtrMYB074-PtrWRKY19 dimers are required to transactive PtrbHLH186. Overexpressing PtrbHLH186 in P. trichocarpa resulted in retarded plant growth, increased guaiacyl lignin, a higher proportion of smaller stem vessels and strong drought-tolerant phenotypes. Knowledge of the PtrMYB074-PtrWRKY19-PtrbHLH186 regulation may help design genetic controls of optimal growth and wood formation to maximize beneficial wood properties while minimizing negative effects on growth.

A PtrLBD39-mediated transcriptional network regulates tension wood formation in Populus trichocarpa.

Tension wood (TW) is a specialized xylem tissue formed in angiosperm trees under gravitational stimulus or mechanical stresses (e.g., bending). The genetic regulation that underlies this important mechanism remains poorly understood. Here, we used laser capture microdissection of stem xylem cells coupled with full transcriptome RNA-sequencing to analyze TW formation in Populus trichocarpa. After tree bending, PtrLBD39 was the most significantly induced transcription factor gene; it has a phylogenetically paired homolog, PtrLBD22. CRISPR-based knockout of PtrLBD39/22 severely inhibited TW formation, reducing cellulose and increasing lignin content. Transcriptomic analyses of CRISPR-based PtrLBD39/22 double mutants showed that these two genes regulate a set of TW-related genes. Chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify direct targets of PtrLBD39. We integrated transcriptomic analyses and ChIP-seq assays to construct a transcriptional regulatory network (TRN) mediated by PtrLBD39. In this TRN, PtrLBD39 directly regulates 26 novel TW-responsive transcription factor genes. Our work suggests that PtrLBD39 and PtrLBD22 specifically control TW formation by mediating a TW-specific TRN in Populus.

Plant biomass fermentation by the extreme thermophile Caldicellulosiruptor bescii for co-production of green hydrogen and acetone: Technoeconomic analysis.

A variety of chemical and biological processes have been proposed for conversion of sustainable low-cost feedstocks into industrial products. Here, a biorefinery concept is formulated, modeled, and analyzed in which a naturally (hemi)cellulolytic and extremely thermophilic bacterium, Caldicellulosiruptor bescii, is metabolically engineered to convert the carbohydrate content of lignocellulosic biomasses (i.e., soybean hulls, transgenic poplar) into green hydrogen and acetone. Experimental validation of C. bescii fermentative performance demonstrated 82% carbohydrate solubilization of soybean hulls and 55% for transgenic poplar. A detailed technical design, including equipment specifications, provides the basis for an economic analysis that establishes metabolic engineering targets. This robust industrial process leveraging metabolically engineered C. bescii yields 206 kg acetone and 25 kg H2 per metric ton of soybean hull, or 174 kg acetone and 21 kg H2 per metric ton transgenic poplar. Beyond this specific case, the model demonstrates industrial feasibility and economic advantages of thermophilic fermentation.

Enzyme Complexes of Ptr4CL and PtrHCT Modulate Co-enzyme A Ligation of Hydroxycinnamic Acids for Monolignol Biosynthesis in Populus trichocarpa.

Co-enzyme A (CoA) ligation of hydroxycinnamic acids by 4-coumaric acid:CoA ligase (4CL) is a critical step in the biosynthesis of monolignols. Perturbation of 4CL activity significantly impacts the lignin content of diverse plant species. In Populus trichocarpa, two well-studied xylem-specific Ptr4CLs (Ptr4CL3 and Ptr4CL5) catalyze the CoA ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. Subsequently, two 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) mediate the conversion of 4-coumaroyl-CoA to caffeoyl-CoA. Here, we show that the CoA ligation of 4-coumaric and caffeic acids is modulated by Ptr4CL/PtrHCT protein complexes. Downregulation of PtrHCTs reduced Ptr4CL activities in the stem-differentiating xylem (SDX) of transgenic P. trichocarpa. The Ptr4CL/PtrHCT interactions were then validated in vivo using biomolecular fluorescence complementation (BiFC) and protein pull-down assays in P. trichocarpa SDX extracts. Enzyme activity assays using recombinant proteins of Ptr4CL and PtrHCT showed elevated CoA ligation activity for Ptr4CL when supplemented with PtrHCT. Numerical analyses based on an evolutionary computation of the CoA ligation activity estimated the stoichiometry of the protein complex to consist of one Ptr4CL and two PtrHCTs, which was experimentally confirmed by chemical cross-linking using SDX plant protein extracts and recombinant proteins. Based on these results, we propose that Ptr4CL/PtrHCT complexes modulate the metabolic flux of CoA ligation for monolignol biosynthesis during wood formation in P. trichocarpa.

CRISPR-Cas9 editing of CAFFEOYL SHIKIMATE ESTERASE 1 and 2 shows their importance and partial redundancy in lignification in Populus tremula × P. alba.

Lignins are cell wall-located aromatic polymers that provide strength and hydrophobicity to woody tissues. Lignin monomers are synthesized via the phenylpropanoid pathway, wherein CAFFEOYL SHIKIMATE ESTERASE (CSE) converts caffeoyl shikimate into caffeic acid. Here, we explored the role of the two CSE homologs in poplar (Populus tremula × P. alba). Reporter lines showed that the expression conferred by both CSE1 and CSE2 promoters is similar. CRISPR-Cas9-generated cse1 and cse2 single mutants had a wild-type lignin level. Nevertheless, CSE1 and CSE2 are not completely redundant, as both single mutants accumulated caffeoyl shikimate. In contrast, the cse1 cse2 double mutants had a 35% reduction in lignin and associated growth penalty. The reduced-lignin content translated into a fourfold increase in cellulose-to-glucose conversion upon limited saccharification. Phenolic profiling of the double mutants revealed large metabolic shifts, including an accumulation of p-coumaroyl, 5-hydroxyferuloyl, feruloyl and sinapoyl shikimate, in addition to caffeoyl shikimate. This indicates that the CSEs have a broad substrate specificity, which was confirmed by in vitro enzyme kinetics. Taken together, our results suggest an alternative path within the phenylpropanoid pathway at the level of the hydroxycinnamoyl-shikimates, and show that CSE is a promising target to improve plants for the biorefinery.

Transcriptional reprogramming of xylem cell wall biosynthesis in tension wood.

Tension wood (TW) is a specialized xylem tissue developed under mechanical/tension stress in angiosperm trees. TW development involves transregulation of secondary cell wall genes, which leads to altered wood properties for stress adaptation. We induced TW in the stems of black cottonwood (Populus trichocarpa, Nisqually-1) and identified two significantly repressed transcription factor (TF) genes: class B3 heat-shock TF (HSFB3-1) and MYB092. Transcriptomic analysis and chromatin immunoprecipitation (ChIP) were used to identify direct TF-DNA interactions in P. trichocarpa xylem protoplasts overexpressing the TFs. This analysis established a transcriptional regulatory network in which PtrHSFB3-1 and PtrMYB092 directly activate 8 and 11 monolignol genes, respectively. The TF-DNA interactions were verified for their specificity and transactivator roles in 35 independent CRISPR-based biallelic mutants and overexpression transgenic lines of PtrHSFB3-1 and PtrMYB092 in P. trichocarpa. The gene-edited trees (mimicking the repressed PtrHSFB3-1 and PtrMYB092 under tension stress) have stem wood composition resembling that of TW during normal growth and under tension stress (i.e., low lignin and high cellulose), whereas the overexpressors showed an opposite effect (high lignin and low cellulose). Individual overexpression of the TFs impeded lignin reduction under tension stress and restored high levels of lignin biosynthesis in the TW. This study offers biological insights to further uncover how metabolism, growth, and stress adaptation are coordinately regulated in trees.

Thermophilic microbial deconstruction and conversion of natural and transgenic lignocellulose.

The potential to convert renewable plant biomasses into fuels and chemicals by microbial processes presents an attractive, less environmentally intense alternative to conventional routes based on fossil fuels. This would best be done with microbes that natively deconstruct lignocellulose and concomitantly form industrially relevant products, but these two physiological and metabolic features are rarely and simultaneously observed in nature. Genetic modification of both plant feedstocks and microbes can be used to increase lignocellulose deconstruction capability and generate industrially relevant products. Separate efforts on plants and microbes are ongoing, but these studies lack a focus on optimal, complementary combinations of these disparate biological systems to obtain a convergent technology. Improving genetic tools for plants have given rise to the generation of low-lignin lines that are more readily solubilized by microorganisms. Most focus on the microbiological front has involved thermophilic bacteria from the genera Caldicellulosiruptor and Clostridium, given their capacity to degrade lignocellulose and to form bio-products through metabolic engineering strategies enabled by ever-improving molecular genetics tools. Bioengineering plant properties to better fit the deconstruction capabilities of candidate consolidated bioprocessing microorganisms has potential to achieve the efficient lignocellulose deconstruction needed for industrial relevance.

A multiscale model of lignin biosynthesis for predicting bioenergy traits in Populus trichocarpa.

Understanding the mechanisms behind lignin formation is an important research area with significant implications for the bioenergy and biomaterial industries. Computational models are indispensable tools for understanding this complex process. Models of the monolignol pathway in Populus trichocarpa and other plants have been developed to explore how transgenic modifications affect important bioenergy traits. Many of these models, however, only capture one level of biological organization and are unable to capture regulation across multiple biological scales. This limits their ability to predict how gene modification strategies will impact lignin and other wood properties. While the first multiscale model of lignin biosynthesis in P. trichocarpa spanned the transcript, protein, metabolic, and phenotypic layers, it did not account for cross-regulatory influences that could impact abundances of untargeted monolignol transcripts and proteins. Here, we present a multiscale model incorporating these cross-regulatory influences for predicting lignin and wood traits from transgenic knockdowns of the monolignol genes. The three main components of this multiscale model are (1) a transcript-protein model capturing cross-regulatory influences, (2) a kinetic-based metabolic model, and (3) random forest models relating the steady state metabolic fluxes to 25 physical traits. We demonstrate that including the cross-regulatory behavior results in smaller predictive error for 23 of the 25 traits. We use this multiscale model to explore the predicted impact of novel combinatorial knockdowns on key bioenergy traits, and identify the perturbation of PtrC3H3 and PtrCAld5H1&2 monolignol genes as a candidate strategy for increasing saccharification efficiencies while reducing negative impacts on wood density and height.

Clinicopathological features and outcome of esophageal neuroendocrine tumor: A retrospective multicenter survey by the digestive endoscopy society of Taiwan.

BACKGROUND & AIMS: Esophageal neuroendocrine tumors (NET) are very rare and mostly carcinomic, carrying poor prognosis. There is still no guideline or consensus on the treatment for esophageal NET. METHODS: Patients with histologically-proven esophageal neuroendocrine tumor were recruited from 9 hospitals in Taiwan between 2002 and 2017. Clinical, laboratory, radiological, endoscopic, pathological data, treatment strategies, follow-up periods, and survivals were collected retrospectively. RESULTS: In total, 39 esophageal NET were analyzed and 38 were neuroendocrine carcinoma (NEC). Sixteen (41%) patients had mixed components with either adenocarcinoma (N = 9, 23%) or squamous-cell carcinoma (SCC) (N = 7, 18%). 64.1% of the patients experienced dysphagia and ulcerative mass was the most comment endoscopic finding. There was a higher proportion of drinkers (54.1%), betel chewers (21.6%) and smokers (64.9%) among the patients than in the general population in Taiwan. Five patients (12.8%) had been diagnosed with other cancers. Definite chemoradiotherapy (N = 14, 35.9%) and surgery (N = 7, 17.9%) were the major treatment. Patients with Ki-67% above the median level (50%) in the tumors tended to have worse survival (P = 0.06). However, presence of mixed component was not a significant survival predictor in our study (P = 0.56). CONCLUSION: Mixed component of an esophageal NET is commonly observed. Staged workup and the principle of treatment can follow that for the common cancer type of esophagus. The risk factors and behaviors of esophageal NEC in Taiwan seem to be similar to that of esophageal SCC.

MYB Transcription Factor161 Mediates Feedback Regulation of Secondary wall-associated NAC-Domain1 Family Genes for Wood Formation.

Wood formation is a complex process that involves cell differentiation, cell expansion, secondary wall deposition, and programmed cell death. We constructed a four-layer wood formation transcriptional regulatory network (TRN) in Populus trichocarpa (black cottonwood) that has four Secondary wall-associated NAC-Domain1 (PtrSND1) transcription factor (TF) family members as the top-layer regulators. We characterized the function of a MYB (PtrMYB161) TF in this PtrSND1-TRN, using transgenic P trichocarpa cells and whole plants. PtrMYB161 is a third-layer regulator that directly transactivates five wood formation genes. Overexpression of PtrMYB161 in P. trichocarpa (OE-PtrMYB161) led to reduced wood, altered cell type proportions, and inhibited growth. Integrative analysis of wood cell-based chromatin-binding assays with OE-PtrMYB161 transcriptomics revealed a feedback regulation system in the PtrSND1-TRN, where PtrMYB161 represses all four top-layer regulators and one second-layer regulator, PtrMYB021, possibly affecting many downstream TFs in, and likely beyond, the TRN, to generate the observed phenotypic changes. Our data also suggested that the PtrMYB161's repressor function operates through interaction of the base PtrMYB161 target-binding system with gene-silencing cofactors. PtrMYB161 protein does not contain any known negative regulatory domains. CRISPR-based mutants of PtrMYB161 in P. trichocarpa exhibited phenotypes similar to the wild type, suggesting that PtrMYB161's activator functions are redundant among many TFs. Our work demonstrated that PtrMYB161 binds to multiple sets of target genes, a feature that allows it to function as an activator as well as a repressor. The balance of the two functions may be important to the establishment of regulatory homeostasis for normal growth and development.

Regulation of Lignin Biosynthesis by Post-translational Protein Modifications.

Post-translational modification of proteins exerts essential roles in many biological processes in plants. The function of these chemical modifications has been extensively characterized in many physiological processes, but how these modifications regulate lignin biosynthesis for wood formation remained largely unknown. Over the past decade, post-translational modification of several proteins has been associated with lignification. Phosphorylation, ubiquitination, glycosylation, and S-nitrosylation of transcription factors, monolignol enzymes, and peroxidases were shown to have primordial roles in the regulation of lignin biosynthesis. The main discoveries of post-translational modifications in lignin biosynthesis are discussed in this review.

Modeling cross-regulatory influences on monolignol transcripts and proteins under single and combinatorial gene knockdowns in Populus trichocarpa.

Accurate manipulation of metabolites in monolignol biosynthesis is a key step for controlling lignin content, structure, and other wood properties important to the bioenergy and biomaterial industries. A crucial component of this strategy is predicting how single and combinatorial knockdowns of monolignol specific gene transcripts influence the abundance of monolignol proteins, which are the driving mechanisms of monolignol biosynthesis. Computational models have been developed to estimate protein abundances from transcript perturbations of monolignol specific genes. The accuracy of these models, however, is hindered by their inability to capture indirect regulatory influences on other pathway genes. Here, we examine the manifestation of these indirect influences on transgenic transcript and protein abundances, identifying putative indirect regulatory influences that occur when one or more specific monolignol pathway genes are perturbed. We created a computational model using sparse maximum likelihood to estimate the resulting monolignol transcript and protein abundances in transgenic Populus trichocarpa based on targeted knockdowns of specific monolignol genes. Using in-silico simulations of this model and root mean square error, we showed that our model more accurately estimated transcript and protein abundances, in comparison to previous models, when individual and families of monolignol genes were perturbed. We leveraged insight from the inferred network structure obtained from our model to identify potential genes, including PtrHCT, PtrCAD, and Ptr4CL, involved in post-transcriptional and/or post-translational regulation. Our model provides a useful computational tool for exploring the cascaded impact of single and combinatorial modifications of monolignol specific genes on lignin and other wood properties.

Use of the lignocellulose-degrading bacterium Caldicellulosiruptor bescii to assess recalcitrance and conversion of wild-type and transgenic poplar.

BACKGROUND: Biological conversion of lignocellulosic biomass is significantly hindered by feedstock recalcitrance, which is typically assessed through an enzymatic digestion assay, often preceded by a thermal and/or chemical pretreatment. Here, we assay 17 lines of unpretreated transgenic black cottonwood (Populus trichocarpa) utilizing a lignocellulose-degrading, metabolically engineered bacterium, Caldicellulosiruptor bescii. The poplar lines were assessed by incubation with an engineered C. bescii strain that solubilized and converted the hexose and pentose carbohydrates to ethanol and acetate. The resulting fermentation titer and biomass solubilization were then utilized as a measure of biomass recalcitrance and compared to data previously reported on the transgenic poplar samples. RESULTS: Of the 17 transgenic poplar lines examined with C. bescii, a wide variation in solubilization and fermentation titer was observed. While the wild type poplar control demonstrated relatively high recalcitrance with a total solubilization of only 20% and a fermentation titer of 7.3 mM, the transgenic lines resulted in solubilization ranging from 15 to 79% and fermentation titers from 6.8 to 29.6 mM. Additionally, a strong inverse correlation (R 2 = 0.8) between conversion efficiency and lignin content was observed with lower lignin samples more easily converted and solubilized by C. bescii. CONCLUSIONS: Feedstock recalcitrance can be significantly reduced with transgenic plants, but finding the correct modification may require a large sample set to identify the most advantageous genetic modifications for the feedstock. Utilizing C. bescii as a screening assay for recalcitrance, poplar lines with down-regulation of coumarate 3-hydroxylase 3 (C3H3) resulted in the highest degrees of solubilization and conversion by C. bescii. One such line, with a growth phenotype similar to the wild-type, generated more than three times the fermentation products of the wild-type poplar control, suggesting that excellent digestibility can be achieved without compromising fitness of the tree.