Genetic variation of biomass recalcitrance in a natural Salix viminalis (L.) population

Background: Salix spp. are high-productivity crops potentially used for lignocellulosic biofuels such as bioethanol. In general, pretreatment is needed to facilitate the enzymatic depolymerization process. Biomass resistance to degradation, i.e., biomass recalcitrance, is a trait which can be assessed by measuring the sugar released after combined pretreatment and enzymatic hydrolysis. We have examined genetic parameters of enzymatic sugar release and other traits related to biorefnery use in a population of 286 natural Salix viminalis clones. Furthermore, we have evaluated phenotypic and genetic correlations between these traits and performed a genomewide association mapping analysis using a set of 19,411 markers. Results: Sugar release (glucose and xylose) after pretreatment and enzymatic saccharifcation proved highly variable with large genetic and phenotypic variations, and chip heritability estimates (h2 ) of 0.23–0.29. Lignin syringyl/guaiacyl (S/G) ratio and wood density were the most heritable traits (h2=0.42 and 0.59, respectively). Sugar release traits were positively correlated, phenotypically and genetically, with biomass yield and lignin S/G ratio. Association mapping revealed seven marker–trait associations below a suggestive signifcance threshold, including one marker associated with glucose release. Conclusions: We identifed lignin S/G ratio and shoot diameter as heritable traits that could be relatively easily evaluated by breeders, making them suitable proxy traits for developing low-recalcitrance varieties. One marker below the suggestive threshold for marker associations was identifed for sugar release, meriting further investigation while also highlighting the difculties in employing genomewide association mapping for complex trait

High activity CAZyme cassette for improving biomass degradation in thermophiles

Abstract Background Thermophilic microorganisms and their enzymes offer several advantages for industrial application over their mesophilic counterparts. For example, a hyperthermophilic anaerobe, Caldicellulosiruptor bescii, was recently isolated from hot springs in Kamchatka, Siberia, and shown to have very high cellulolytic activity. Additionally, it is one of a few microorganisms being considered as viable candidates for consolidated bioprocessing applications. Moreover, C. bescii is capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This ability is accomplished by the production and secretion of free, multi-modular and multi-functional enzymes, one of which, CbCel9A/Cel48A also known as CelA, is able to outperform enzymes found in commercial enzyme preparations. Furthermore, the complete C. bescii exoproteome is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Therefore, understanding the functional diversity of enzymes in the C. bescii exoproteome and how inter-molecular synergy between them confers C. bescii with its high cellulolytic activity is an important endeavor to enable the production of more efficient biomass degrading enzyme formulations and in turn, better cellulolytic industrial microorganisms. Results To advance the understanding of the C. bescii exoproteome we have expressed, purified, and tested four of the primary enzymes found in the exoproteome and we have found that the combination of three or four of the most highly expressed enzymes exhibit synergistic activity. We also demonstrated that discrete combinations of these enzymes mimic and even  improve upon the activity of the whole C. bescii exoproteome, even though some of the enzymes lack significant activity on their own. Conclusions We have demonstrated that it is possible to replicate the cellulolytic activity of the native C. bescii exoproteome utilizing a minimal gene set, and that these minimal gene sets are more active than the whole exoproteome. In the future, this may lead to more simplified and efficient cellulolytic enzyme preparations or yield improvements when these enzymes are expressed in microorganisms engineered for consolidated bioprocessing

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Additional file 1. Additional tables and figures

Study of traits and recalcitrance reduction of field-grown COMT down-regulated switchgrass

This article discusses the fundamental features of why biomass is recalcitrant to conversion without pretreatment

MOESM1 of Study of traits and recalcitrance reduction of field-grown COMT down-regulated switchgrass

Additional file 1. Supporting figures and tables of understanding the reduced cell wall recalcitrance of field-grown COMT Down-regulated switchgrass. Figure S1. Scheme of proposed lignin biosynthesis involving COMT. Figure S2. Relationship of cellulose DP to glucose release efficiency of unpretreated switchgrass. Figure S3. Relationship of hemicellulose molecular weights to xylose release efficiency of unpretreated switchgrass. Figure S4. Relationship of Cellulose crystallinity (CrI) to glucose release unpretreated switchgrass. Table S1. Original data used for Fig. 1: Chemical composition (per g cell wall residue) of field-grown switchgrass in years 2 and 3. The values reported are the average of 5 biological replicates from each control group, and 10 biological replicates from each transgenic group. Student t test was used as a statistical analysis for the difference between the transgenic and control groups. Table S2. Data used for Fig. 2a and b: Sugar release (mg per g cell wall residues) from hydrothermally pretreated (a) and unpretreated (b) switchgrass in years 2 and 3 after 72 h enzymatic hydrolysis (the value reported is the average of 5 biological replicates from each control group, and 10 biological replicates from each transgenic group). Student t test was used as a statistical analysis for the difference between the transgenic and control groups. Table S3. Original data used for Fig. 2c: the relationship of total sugar (glucose and xylose) release for pretreated switchgrass from enzymatic hydrolysis to lignin content (wt% of cell wall residues). Table S4. Original data used for Fig. 2d: the relationship of total sugar (glucose and xylose) release for unpretreated switchgrass from enzymatic hydrolysis to lignin content (wt% of cell wall residues). Table S5. Original data used for Figs. 3 and 4: distribution of DO adsorption and relationship of DO its relationship to sugar release (Fig. 3). Hemicellulose molecular weight distribution and Cellulose crystallinity index (CrI) (Fig. 4). Student’s t test was used as a statistical analysis for the difference between the transgenic and control groups. Table S6. Student’s t test results of traits difference in field-grown switchgrass between year 2 and 3

Improving wood properties for wood utilization through multi-omics integration in lignin biosynthesis.

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