Lignocellulose, an abundant renewable resource, presents a promising alternative for sustainable energy and industrial applications. However, large-scale adoption of lignocellulosic feedstocks faces considerable obstacles, including scalability, bioprocessing efficiency, and resilience to climate change. This Review examines current efforts and future opportunities for leveraging lignocellulosic feedstocks in bio-based energy and products, with a focus on enhancing conversion efficiency and scalability. It also explores emerging biotechnologies such as CRISPR-based genome editing informed by machine learning, aimed at improving feedstock traits and reducing the environmental impact of fossil fuel dependence.

Plants develop tolerance to drought by activating genes with altered levels of epigenetic modifications. Specific transcription factors are involved in this activation, but the molecular connections within the regulatory system are unclear. Here, we analyzed genome-wide acetylated lysine residue 9 of histone H3 (H3K9ac) enrichment and examined its association with transcriptomes in Populus trichocarpa under drought stress. We revealed that abscisic acid-Responsive Element (ABRE) motifs in promoters of the drought-responsive genes PtrNAC006, PtrNAC007, and PtrNAC120 are involved in H3K9ac enhancement and activation of these genes. Overexpressing these PtrNAC genes in P. trichocarpa resulted in strong drought-tolerance phenotypes. We showed that the ABRE binding protein PtrAREB1-2 binds to ABRE motifs associated with these PtrNAC genes and recruits the histone acetyltransferase unit ADA2b-GCN5, forming AREB1-ADA2b-GCN5 ternary protein complexes. Moreover, this recruitment enables GCN5-mediated histone acetylation to enhance H3K9ac and enrich RNA polymerase II specifically at these PtrNAC genes for the development of drought tolerance. CRISPR editing or RNA interference-mediated downregulation of any of the ternary members results in highly drought-sensitive P. trichocarpa. Thus, the combinatorial function of the ternary proteins establishes a coordinated histone acetylation and transcription factor-mediated gene activation for drought response and tolerance in Populus species.

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.

Wood remains the world’s most abundant and renewable resource for timber and pulp and is an alternative to fossil fuels. Understanding the molecular regulation of wood formation can advance the engineering of wood for more efficient material and energy productions. We integrated a black cottonwood (Populus trichocarpa) wood-forming cell system with quantitative transcriptomics and chromatin binding assays to construct a transcriptional regulatory network (TRN) directed by a key transcription factor (TF), PtrSND1-B1 (secondary wall-associated NAC-domain protein). The network consists of four layers of TF–target gene interactions with quantitative regulatory effects, describing the specificity of how the regulation is transduced through these interactions to activate cell wall genes (effector genes) for wood formation. PtrSND1-B1 directs 57 TF–DNA interactions through 17 TFs transregulating 27 effector genes. Of the 57 interactions, 55 are novel. We tested 42 of these 57 interactions in 30 genotypes of transgenic P. trichocarpa and verified that ∼90% of the tested interactions function in vivo. The TRN reveals common transregulatory targets for distinct TFs, leading to the discovery of nine TF protein complexes (dimers and trimers) implicated in regulating the biosynthesis of specific types of lignin. Our work suggests that wood formation may involve regulatory homeostasis determined by combinations of TF–DNA and TF–TF (protein–protein) regulations.

We established a predictive kinetic metabolic-flux model for the 21 enzymes and 24 metabolites of the monolignol biosynthetic pathway using Populus trichocarpa secondary differentiating xylem. To establish this model, a comprehensive study was performed to obtain the reaction and inhibition kinetic parameters of all 21 enzymes based on functional recombinant proteins. A total of 104 Michaelis-Menten kinetic parameters and 85 inhibition kinetic parameters were derived from these enzymes. Through mass spectrometry, we obtained the absolute quantities of all 21 pathway enzymes in the secondary differentiating xylem. This extensive experimental data set, generated from a single tissue specialized in wood formation, was used to construct the predictive kinetic metabolic-flux model to provide a comprehensive mathematical description of the monolignol biosynthetic pathway. The model was validated using experimental data from transgenic P. trichocarpa plants. The model predicts how pathway enzymes affect lignin content and composition, explains a long-standing paradox regarding the regulation of monolignol subunit ratios in lignin, and reveals novel mechanisms involved in the regulation of lignin biosynthesis. This model provides an explanation of the effects of genetic and transgenic perturbations of the monolignol biosynthetic pathway in flowering plants.

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.