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Redesigning plant cell walls for the biomass-based bioeconomy

Open AccessPublished:August 31, 2020DOI:https://doi.org/10.1074/jbc.REV120.014561
      Lignocellulosic biomass—the lignin, cellulose, and hemicellulose that comprise major components of the plant cell well—is a sustainable resource that could be utilized in the United States to displace oil consumption from heavy vehicles, planes, and marine-going vessels and commodity chemicals. Biomass-derived sugars can also be supplied for microbial fermentative processing to fuels and chemicals or chemically deoxygenated to hydrocarbons. However, the economic value of biomass might be amplified by diversifying the range of target products that are synthesized in living plants. Genetic engineering of lignocellulosic biomass has previously focused on changing lignin content or composition to overcome recalcitrance, the intrinsic resistance of cell walls to deconstruction. New capabilities to remove lignin catalytically without denaturing the carbohydrate moiety have enabled the concept of the “lignin-first” biorefinery that includes high-value aromatic products. The structural complexity of plant cell-wall components also provides substrates for polymeric and functionalized target products, such as thermosets, thermoplastics, composites, cellulose nanocrystals, and nanofibers. With recent advances in the design of synthetic pathways, lignocellulosic biomass can be regarded as a substrate at various length scales for liquid hydrocarbon fuels, chemicals, and materials. In this review, we describe the architectures of plant cell walls and recent progress in overcoming recalcitrance and illustrate the potential for natural or engineered biomass to be used in the emerging bioeconomy.
      From the origin of our species, plant cell walls have provided food, animal feed, and other fundamental resources to support human existence. Woody biomass is used for building materials and fine furniture; natural fibers for clothes, textiles, and paper products; and biomass for solid fuel. Economic activity resulting from these and other uses is the basis of a traditional bioeconomy. Whereas climate change is an existential threat to agricultural production for the traditional bioeconomy, mitigation of the greenhouse gas emissions that cause climate change could be accomplished through a vibrant and emerging biomass-based bioeconomy, enabled by recombinant DNA technologies (
      National Academies of ScienceEngineering, and Medicine
      ). Oil is the predominant source of transportation fuel, and petrochemicals provide the starting materials for tens of thousands of synthetic chemicals and materials. As a matter of urgency (
      Intergovernmental Panel on Climate Change
      ), we now need to shift from oil, derived from long-dead organisms, to living organisms that can provide chemicals, fuels, and materials.
      The diversity of plant metabolism, natural and engineered, provides a foundation for engineering biology to create economic value. Living plant cells synthesize between 100,000 and 1 million kinds of molecules (
      • Fang C.
      • Fernie A.R.
      • Luo J.
      Exploring the diversity of plant metabolism.
      ). Some natural plant products have nutritional or pharmaceutical value and form the basis of foods, supplements, and drugs (
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      Engineering complex metabolic pathways in plants.
      ). Others govern interactions of the plant with its environment, contributing to sustainable food and feed production (
      • Ferreyra M.L.F.
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      Flavonoids: biosynthesis, biological functions, and biotechnological applications.
      ). A semisynthetic pathway to produce substrates for the antimalarial artemisinin has been engineered into Escherichia coli (
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      • Eachus R.A.
      • Trieu W.
      • Ro D.K.
      • Keasling J.D.
      Engineering Escherichia coli for production of functionalized terpenoids using plant P450s.
      ). The Z-MAPP vaccine, effective against Ebola, comprised recombinant antibodies produced in tobacco plants (
      • Qiu X.
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      Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp.
      ). Capabilities in genetic engineering and synthetic biology could be used to produce a large share of the global economy's physical inputs in manufacturing: novel materials of improved quality, designed to be recyclable after use and produced in a way that reduces greenhouse gas emissions (
      • Chui M.
      • Evers M.
      • Manyika J.
      • Zheng A.
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      ). From analysis of 400 potential applications across the life sciences, the McKinsey Global Institute estimated an economic impact of $2–4 trillion over the next 2 decades (
      • Chui M.
      • Evers M.
      • Manyika J.
      • Zheng A.
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      ).
      Most high-value products of plant secondary metabolism are produced in tiny amounts. To address how plants can displace a significant proportion of oil consumption requires use of the major products of primary metabolism, sugars and amino acids. The United States has an annually sustainable resource of over 1 billion tons of lignocellulosic biomass (
      United States Department of Energy
      ). Glucose is the carbon source in fermentations and a major cost to companies using microbial synthetic biology: deconstruction of cellulose could provide an abundant source of sugar. Sugars and aromatics derived from plant cell walls can be incorporated or catalytically transformed into fuels and chemicals (
      • McCann M.C.
      • Carpita N.C.
      Biomass recalcitrance: a multi-scale, multi-factor and conversion-specific property.
      ). Decades of research have overcome the technological barriers to production of cellulose-derived glucose for fermentation to ethanol. However, a cellulosic ethanol industry has not developed because of the low cost of petroleum. In the United States, hydraulic fracturing (fracking) of microporous shale has increased domestic oil and gas production such that the country has became a net exporter, and the price of a barrel of oil, set by OPEC, has dropped to historically low levels. Electric and hybrid vehicles powered by renewable energy sources are becoming viable long-term options for light ground transportation (
      United States Department of Energy
      ). Thus, the range of products, and their economic value, derived from lignocellulosic biomass needs to expand to gain the societal benefits of combatting climate change. To offset the 60% of the barrel of oil that is not used for gasoline, these products should include liquid hydrocarbon fuels and commodity chemicals (
      United States Department of Energy
      ). Air, marine, and heavy-duty modes of transportation, which contribute one-third of United States transportation greenhouse gas emissions, will remain dependent upon energy-dense, liquid hydrocarbon fuels for decades because of slow fleet turnover. For example, new aircraft typically have production runs that last for 10–15 years and a service lifetime of 25–30 years. Analogous to the dried distillers' grains produced as animal feed in ethanol biorefineries, higher-value co-products from biomass are needed to make biorefineries economically viable (
      United States Department of Energy
      ).
      In this review, we describe the architectures of plant cell walls and recent progress in overcoming recalcitrance, the intrinsic resistance of cell walls to deconstruction of their component polymers to sugars and aromatics (
      • McCann M.C.
      • Carpita N.C.
      Biomass recalcitrance: a multi-scale, multi-factor and conversion-specific property.
      ,
      • Himmel M.E.
      • Ding S.Y.
      • Johnson D.K.
      • Adney W.S.
      • Nimlos M.R.
      • Brady J.W.
      • Foust T.D.
      Biomass recalcitrance: engineering plants and enzymes for biofuels production.
      ). Recalcitrance gives rise to the differences between theoretical yields of sugars and aromatics from cell walls and actual yields recovered from their enzymatic or chemical deconstruction. In particular, the ability to deconstruct lignin into aromatic monomers has changed the concept of the cellulosic biorefinery. We discuss how biomass-derived sugars and aromatics can be used as substrates for conversion to hydrocarbon fuels or as monomers for polymerization into plastics. The main scaffolding component of cell walls, cellulose, can be isolated as nanocrystals or nanofibers and incorporated into materials with novel properties. Polysaccharides extracted from cell walls are used to coat cellulose, modifying its biophysical properties. These examples are not intended to be comprehensive but to illustrate the potential for natural or engineered biomass to be used in the emerging bioeconomy. We outline some future directions of research needed to fulfill this potential.

      Plant cell-wall architectures

      Plant cell walls are the largest stable reservoir of carbon fixed by photosynthesis. The cell walls of all flowering plants contain cellulose microfibrils (Fig. 1). Each microfibril comprises 18–24 (1→4)-β-d-glucan chains, hydrogen-bonded to each other to form scaffolding cables of 2.3–3 nm in diameter (
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      ,
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      ,
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      Structure of cellulose microfibrils in primary cell walls from collenchyma.
      ). These diameters are consistent with the size and spacing of the six-particle “rosette complexes” that synthesize cellulose microfibrils (
      • Giddings Jr., T.H.
      • Brower D.L.
      • Staehelin L.A.
      Visualization of particle complexes in the plasma membrane of Micrasterias denticulata associated with the formation of cellulose fibrils in primary and secondary cell walls.
      ). In cotton fiber cellulose, each glucan chain contains 10,000–14,000 glucosyl units (
      • Brown R.M.
      The control of molecular weight and molecular-weight distribution in the biogenesis of cellulose.
      ,
      • Timpa J.D.
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      Analysis of cell-wall polymers during cotton fiber development.
      ), with corresponding lengths of 5–7 μm (
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      On the determination of crystallinity and cellulose content in plant fibres.
      ). However, the individual chains begin and end at different points within the microfibril, such that individual microfibrils might extend several millimeters. Cellulose microfibrils in wood are packed into dense macrofibrils of much larger size, from 10 to 20 nm to as high as 60 nm in diameter (
      • Donaldson L.
      Cellulose microfibril aggregates and their size variation with cell wall type.
      ). Electron tomography (
      • Xu P.
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      • Staehelin L.A.
      Dual-axis electron tomography: a new approach for investigating the spatial organization of wood cellulose microfibrils.
      ) and atomic force microscopy (
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      Pore and matrix distribution in the fiber wall revealed by atomic force microscopy and image analysis.
      ) showed macrofibrils to be interspersed with other polymers. The intrinsic crystallinity of each microfibril and the extent of bundling of microfibrils into macrofibrils both contribute to recalcitrance. How cellulose synthase complexes form cellulose microfibrils at the plant cell surface and how microfibrils coalesce into macrofibrils is not known.
      Figure thumbnail gr1
      Figure 1The structure of the plant cell wall. A, the primary cell walls synthesized during growth are constructed with a scaffold of cellulose microfibrils cross-linked by hemicelluloses and embedded in a co-extensive matrix of pectic polysaccharides and some hemicelluloses. Pectins comprise two fundamental polymers, homogalacturonan and rhamnogalacturonan I (RG-I), and the RG-I is branched with 5-arabinans and type I arabinogalactans. In the type I wall found in all dicots and certain monocots, xyloglucan is the major hemicellulose, and the matrix is largely pectin. In the type II wall of grasses and related monocots, glucuronoarabinoxylan (GAX) is the major hemicellulose, and the matrix is largely GAX. Grasses uniquely contain (1→3),(1→4)-β-d-glucan hemicellulose, and both types of walls contain small amounts of glucomannan tightly adherent to cellulose. Also unique to the type II wall is a phenylpropanoid network (in red). Adapted from Ref.
      • Carpita N.C.
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      Maize and sorghum: genetic resources for bioenergy grasses.
      . B, fluorescence image of a cross-section of developing wood in the stem of Pinus radiata. The compound middle lamella (CML) fuses with the primary wall and is the initial site of lignin polymerization. A secondary cell wall (SCW) is elaborated with the compound middle lamella. Bar, 20 μm. Adapted from Ref.
      • Donaldson L.A.
      Lignification and lignin topochemistry—an ultrastructural view.
      . C, model of the secondary cell wall. Cellulose microfibrils are coated with xylan and mannan and embedded in a co-extensive lignin matrix. Adapted from Ref.
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      .
      In primary (growing) walls of most flowering plants, xyloglucans are the predominant hemicelluloses that tether cellulose microfibrils into a network, embedded in a second network of pectins (Fig. 1A). Pectins comprise a class of acidic polysaccharides, including homogalacturonans, polymers of galacturonic acid, and rhamnogalacturonan-I, a pectic polymer of alternating rhamnose and galacturonic acid residues substituted with side chains of arabinans and galactans (
      • Yang H.
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      Rhamnogalacturonan I is a determinant of cell-cell adhesion in poplar wood.
      ). In grasses and related monocot species, including bioenergy crops, such as sorghum and switchgrass, glucuronoarabinoxylans are the most abundant hemicelluloses that, together with cross-linking phenylpropanoid networks, form the primary walls (Fig. 1A).
      In certain cell types, thickened and lignified secondary wall composites of cellulose and glucuronoxylans and other hemicelluloses, interconnected with a network of acidic phenylpropanoids and lignin, are elaborated within primary walls (Fig. 1B). These walls contribute disproportionately to harvested biomass (
      • Chanoca A.
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      Lignin engineering in forest trees.
      ,
      • Ralph J.
      • Lapierre C.
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      Lignin structure and its engineering.
      ). The lignin heteropolymer is produced via the oxidative coupling of monolignol subunits corresponding to p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) lignins (Fig. 2). Monolignols are synthesized by several enzymatic reactions, beginning with deamination of phenylalanine to trans-cinnamic acid by the ammonia lyase (PAL), subsequent hydroxylation to p-coumaric acid cinnamate 4-hydroxylase (C4H), and its coupling to CoA by 4-coumarate:CoA ligase (4CL) (Fig. 2). Pathways can then involve coupling to shikimate derivatives, 3′-hydroxylation and methoxylation to ferulic acid, and conversion of the acid to aldehyde by a CoA-dependent cinnamoyl reductase (CCR). Hydroxylation by ferulate 5-hydroxylase (F5H), subsequent methoxylation by caffeic acid O-methyltransferase (COMT), and conversion of the aldehydes to alcohols by cinnamyl alcohol dehydrogenase (CAD) yields the three principal components of lignin as H, G, and S units (Fig. 2). These monolignols, synthesized in the cytoplasm, are transported across the plasma membrane into the wall, where they are polymerized to form lignin macromolecules by free radical–mediated mechanisms (
      • Ralph J.
      • Lapierre C.
      • Boerjan W.
      Lignin structure and its engineering.
      ). Lignin composition is therefore determined by the relative proportions of the exported monolignols and other aromatics and the different kinds of covalent linkages that can be formed between them (
      • Chanoca A.
      • de Vries L.
      • Boerjan W.
      Lignin engineering in forest trees.
      ,
      • Ralph J.
      • Lapierre C.
      • Boerjan W.
      Lignin structure and its engineering.
      ). Because lignin is structurally heterogeneous, it is difficult to deconstruct to yield intact aromatic molecules as reaction products.
      Figure thumbnail gr2
      Figure 2The monolignol biosynthesis pathways. Synthesis begins with deamination of phenylalanine by the PAL, to trans-cinnamic acid, or by deamination of tyrosine to p-coumaric acid by tyrosine ammonia lyase (TAL). A C4H converts trans-cinnamic acid to p-coumaric acid, which can be converted to caffeic acid by a p-coumaroyl quinate/shikimate 3′hydroxylase (C3′H) and subsequently converted to ferulic acid by a COMT. However, the typical route to monolignols is by the formation of p-coumaroyl-CoA by 4CL and to feruloyl-CoA by sequential reactions of a p-hydroxycinnamoyl-CoA:quinate/shikimate-p-hydroxycinnamoyltransferase (HCT), a C3′H, a 4CL, and a caffeoyl-CoA O-methyltransferase (CCoAOMT). The p-coumaroyl-CoA can be directly reduced to p-coumaraldehyde by CCR and to p-coumaryl alcohol by CAD to make the monolignol substrate for H-lignin. CCR and CAD also reduce feruloyl-CoA to coniferaldehyde and to coniferyl alcohol substrate for G-lignin. A combination of F5H and COMT, before or after reduction by CAD, converts coniferaldehyde or coniferyl alcohol to sinapaldehyde or sinapyl alcohol, the substrate of S-lignin. Three important variations in these pathways are the reduction, by CCR and CAD, of caffeoyl-CoA to the caffeyl alcohol substrate of C-lignin and the cycling of ferulic acid into feruloyl-CoA by 4CL and back to ferulic acid by coniferaldehyde dehydrogenase (CALDH). Di- and trimethoxycinnamyl alcohols can also be made by MOMTs. For each of these reactions, the change in molecular structure is indicated in red. Adapted from Ref.
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      , as modified in Ref.
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      Cell-wall compositions vary during plant development, among cell types within a plant, and between species, in response to environmental stimuli (
      • Carpita N.C.
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      The cell wall.
      ). Cell-wall heterogeneity is a challenge in achieving material of consistent composition delivered to the biorefinery. It is also a largely unexploited opportunity to use molecular and genetic technologies to optimize compositions and architectures of different cell types and in different plant organs for end uses. Maximizing the recovery of biomass carbon into biofuels and desired co-products requires flexible design capabilities to produce cell-wall architectures that can be easily and completely deconstructed for current and future conversion processes (
      • McCann M.C.
      • Carpita N.C.
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      ). As robust cell-wall architectures are integral to plant growth and development, trade-offs of tailoring biomass quality for conversion processes and field performance must be addressed so as to not compromise yield or sustainability. A major knowledge gap is how biosynthetic products are integrated into composite structures, how their individual structural complexities contribute from molecular- to macro-scale architectures, and how the rules might be broken to redesign cell-wall architectures for production of high-value products.

      Modulating lignin content and composition to maximize recovery of sugars

      Fermentations of sugars derived from cellulose and xylans produce second-generation biofuels, including ethanol or butanol, in contrast to first-generation ethanol produced from starch-derived glucose (
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      Battle for the barrel.
      ). The second-generation biorefinery depends on optimizing recovery of glucose and xylose from biomass particles (
      • Hayes D.J.
      An examination of biorefining processes, catalysts and challenges.
      ). In this context, biomass recalcitrance has been assumed to be the resistance of plant cell walls to enzymatic hydrolysis (
      • Himmel M.E.
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      • Adney W.S.
      • Nimlos M.R.
      • Brady J.W.
      • Foust T.D.
      Biomass recalcitrance: engineering plants and enzymes for biofuels production.
      ), and the molecular basis of recalcitrance has been attributed to strong interactions between lignin and cellulose, which limit access of saccharifying enzymes to their polymeric substrates (
      • Chen F.
      • Dixon R.A.
      Lignin modification improves fermentable sugar yields for biofuel production.
      ). Many genes encoding the enzymes of lignin biosynthesis have been identified through genetic functional analyses (
      • Chanoca A.
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      Lignin engineering in forest trees.
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      ,
      • Penning B.W.
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      ), enabling engineering of lignin content or composition (
      • Chanoca A.
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      Lignin engineering in forest trees.
      ,
      • Ralph J.
      • Lapierre C.
      • Boerjan W.
      Lignin structure and its engineering.
      ).
      In poplar (Populus spp.), disruption of genes encoding certain enzymes of monolignol synthesis, such as C4H (
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      ), resulted in drastic decreases in lignin content. In some instances, down-regulation of early enzymes in monolignol synthesis was correlated with increased saccharification yields of glucose and xylose (
      • Mansfield S.D.
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      ). Down-regulation of CAD to block conversion to monolignol resulted in only modest reduction of lignin in poplar, but enhanced saccharification (
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      ), constitute the brown-midrib mutations of sorghum that have enhanced digestibility to improve both ruminant nutrition and sugar yield from saccharification (
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      • Rivera L.
      • Ejeta G.
      Allelic association, chemical characterization and saccharification properties of brown midrib mutants of sorghum (Sorghum bicolor (L.) Moench).
      ). Manipulation of the expression of these genes was sometimes accompanied by reduction of biomass yield (
      • Bjurhager I.
      • Olsson A.-M.
      • Zhang B.
      • Gerber L.
      • Kumar M.
      • Berglund L.A.
      • Burgert I.
      • Sundberg B.
      • Salmén L.
      Ultrastructure and mechanical properties of Populus wood with reduced lignin content caused by transgenic down-regulation of cinnamate 4-hydroxylase.
      ,
      • Leplé J.-C.
      • Dauwe R.
      • Morreel K.
      • Storme V.
      • Lapierre C.
      • Pollet B.
      • Naumann A.
      • Kang K.-Y.
      • Kim H.
      • Ruel K.
      • Lefèbvre A.
      • Joseleau J.-P.
      • Grima-Pettenati J.
      • De Rycke R.
      • Andersson-Gunnerås S.
      • et al.
      Downregulation of cinnamoyl-coenzyme A reductase in poplar: multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure.
      ,
      • Van Acker R.
      • Leplé J.-C.
      • Aerts D.
      • Storme V.
      • Goeminne G.
      • Ivens B.
      • Légée F.
      • Lapierre C.
      • Piens K.
      • Van Montagu M.C.E.
      • Santoro N.
      • Foster C.E.
      • Ralph J.
      • Soetaert W.
      • Pilate G.
      • et al.
      Improved saccharification and ethanol yield from field-grown transgenic poplar deficient in cinnamoyl-CoA reductase.
      ,
      • Stout A.T.
      • Davis A.A.
      • Domec J.C.
      • Yang C.M.
      • Shi R.
      • King J.S.
      Growth under field conditions affects lignin content and productivity in transgenic Populus trichocarpa with altered lignin biosynthesis.
      ,
      • Zhou X.
      • Ren S.
      • Lu M.
      • Zhao S.
      • Chen Z.
      • Zhao R.
      • Lv J.
      Preliminary study of cell wall structure and its mechanical properties of C3H and HCT RNAI transgenic poplar sapling.
      ). Such variable losses might be associated with incomplete xylem stiffening that leads to collapse under water stress (
      • Coleman H.D.
      • Park J.-Y.
      • Nair R.
      • Chapple C.
      • Mansfield S.D.
      RNAi-mediated suppression of p-coumaroyl-CoA 3-hydroxylase in hybrid poplar impacts lignin deposition and soluble secondary metabolism.
      ) or accumulation of inhibitory phenolic substances (
      • Muro-Villanueva F.
      • Mao X.
      • Chapple C.
      Linking phenylpropanoid metabolism, lignin deposition, and plant growth inhibition.
      ).
      To improve saccharification yield without compromising growth and biomass quality, alternative strategies took advantage of the plasticity of lignin synthesis. Adjusting the pool of available monolignols by genetic manipulation of enzymes in the biosynthetic pathway can change lignin composition (
      • Muro-Villanueva F.
      • Mao X.
      • Chapple C.
      Linking phenylpropanoid metabolism, lignin deposition, and plant growth inhibition.
      ). Down-regulation of F5H in poplar produced lignin that is enriched in G units at the expense of S units, but overexpression of the gene resulted in more than 90% S-lignin content (
      • Stewart J.J.
      • Akiyama T.
      • Chapple C.
      • Ralph J.
      • Mansfield S.D.
      The effects on lignin structure of overexpression of ferulate 5-hydroxylase in hybrid poplar.
      ,
      • Yang H.
      • Zhang X.
      • Luo H.
      • Liu B.
      • Shiga T.M.
      • Li X.
      • Kim J.I.
      • Rubinelli P.
      • Overton J.C.
      • Subramanyam V.
      • Abu-Omar M.
      • Chapple C.
      • Donohoe B.S.
      • Cooper B.R.
      • Mo H.
      • et al.
      Overcoming cellulose recalcitrance in woody biomass for the lignin-first biorefinery.
      ). This high-S modification enhanced glucose and xylose release without impact on tree growth. A monolignol 4-O-methyltransferase (MOMT) gene overexpressed in poplar resulted in formation of 4-O-methyl coniferyl and sinapyl alcohols that terminated lignin polymerization, reducing lignin content and increasing saccharification yield (
      • Cai Y.
      • Zhang K.
      • Kim H.
      • Hou G.
      • Zhang X.
      • Yang H.
      • Feng H.
      • Miller L.
      • Ralph J.
      • Liu C.-J.
      Enhancing digestibility and ethanol yield of Populus wood via expression of an engineered monolignol 4-O-methyltransferase.
      ). Introducing alkali-cleavable ferulate esters into the lignin chains resulted in fragmentation into smaller oligomers to reduce recalcitrance (
      • Karlen S.D.
      • Zhang C.
      • Peck M.L.
      • Smith R.A.
      • Padmakshan D.
      • Helmich K.E.
      • Free H.C.A.
      • Lee S.
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      • Lu F.
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      • Sibout R.
      • Grabber J.H.
      • Runge T.M.
      • Mysore K.S.
      • et al.
      Monolignol ferulate conjugates are naturally incorporated into plant lignins.
      ).
      Remarkable flexibility exists in the natural and synthetic monomers that can be polymerized into lignin (
      • Ralph J.
      • Lapierre C.
      • Boerjan W.
      Lignin structure and its engineering.
      ). Variants of the classic H-, G-, and S-lignin are observed in nature; for example, caffeyl alcohol (C) is polymerized into the C-lignin of seed coats in vanilla bean and some cacti (
      • Chen F.
      • Tobimatsu Y.
      • Havkin-Frenkel D.
      • Dixon R.A.
      • Ralph J.
      A polymer of caffeyl alcohol in plant seeds.
      ), and 5-hydroxyguaiacyl units are polymerized into 5-hydroxy-G-lignin in other cactus species (
      • Chen F.
      • Tobimatsu Y.
      • Jackson L.
      • Nakashima J.
      • Ralph J.
      • Dixon R.A.
      Novel seed coat lignins in the Cactaceae: structure, distribution and implications for the evolution of lignin diversity.
      ) (Fig. 2). The flavonoid tricin is present in some grass species (
      • Lan W.
      • Rencoret J.
      • Lu F.
      • Karlen S.D.
      • Smith B.G.
      • Harris P.J.
      • del Río J.C.
      • Ralph J.
      Tricin-lignins: occurrence and quantitation of tricin in relation to phylogeny.
      ), and hydroxystilbenes, such as resveratrol, also occur as natural substituents of lignin (
      • del Río J.C.
      • Rencoret J.
      • Gutiérrez A.
      • Kim H.
      • Ralph J.
      Hydroxystilbenes are monomers in palm fruit endocarp lignins.
      ). Mutations in CAD, that block the last step in reduction of hydroxycinnamaldehydes to monolignols, incorporate the aldehydes as well as their respective alcohols (
      • Van Acker R.
      • Déjardin A.
      • Desmet S.
      • Hoengenaert L.
      • Vanholme R.
      • Morreel K.
      • Laurans F.
      • Kim H.
      • Santoro N.
      • Foster C.
      • Goeminne G.
      • Légée F.
      • Lapierre C.
      • Pilate G.
      • Ralph J.
      • et al.
      Different routes for conifer- and sinapaldehyde and higher saccharification upon deficiency in the dehydrogenase CAD1.
      ), and mutations in COMT generate 5-hydroxyguaiacyl units that are readily polymerized into lignin at the expense of S-units (
      • Yang H.
      • Zhang X.
      • Luo H.
      • Liu B.
      • Shiga T.M.
      • Li X.
      • Kim J.I.
      • Rubinelli P.
      • Overton J.C.
      • Subramanyam V.
      • Abu-Omar M.
      • Chapple C.
      • Donohoe B.S.
      • Cooper B.R.
      • Mo H.
      • et al.
      Overcoming cellulose recalcitrance in woody biomass for the lignin-first biorefinery.
      ,
      • Ralph J.
      • Lapierre C.
      • Marita J.M.
      • Kim H.
      • Lu F.
      • Hatfield R.D.
      • Ralph S.
      • Chapple C.
      • Franke R.
      • Hemm M.R.
      • Van Doorsselaere J.
      • Sederoff R.R.
      • O'Malley D.M.
      • Scott J.T.
      • MacKay J.J.
      • et al.
      Elucidation of new structures in lignins of CAD- and COMT-deficient plants by NMR.
      ).
      Not all phenylpropanoids of the monolignol pathway are incorporated in abundance without growth penalty (
      • Wang J.P.
      • Matthews M.L.
      • Williams C.M.
      • Shi R.
      • Yang C.
      • Tunlaya.-Anukit S.
      • Chen H.-C.
      • Li Q.
      • Liu J.
      • Lin C.-Y.
      • Naik P.
      • Sun Y.-H.
      • Loziuk P.L.
      • Yeh T.-F.
      • Kim H.
      • et al.
      Improving wood properties for wood utilization through multi-omics integration in lignin biosynthesis.
      ). Down-regulation of CAD introduced more easily cleavable aldehydes (
      • Van Acker R.
      • Déjardin A.
      • Desmet S.
      • Hoengenaert L.
      • Vanholme R.
      • Morreel K.
      • Laurans F.
      • Kim H.
      • Santoro N.
      • Foster C.
      • Goeminne G.
      • Légée F.
      • Lapierre C.
      • Pilate G.
      • Ralph J.
      • et al.
      Different routes for conifer- and sinapaldehyde and higher saccharification upon deficiency in the dehydrogenase CAD1.
      ). When reduced CAD expression was coupled with overexpression of F5H in Arabidopsis, sinapyl aldehyde content was increased, but plants were severely dwarfed (
      • Anderson N.A.
      • Tobimatsu Y.
      • Ciesielski P.N.
      • Ximenes E.
      • Ralph J.
      • Donohoe B.S.
      • Ladisch M.
      • Chapple C.
      Manipulation of guaiacyl and syringyl monomer synthesis in an Arabidopsis cinnamyl alcohol dehydrogenase mutant results in atypical lignin biosynthesis and modified cell wall structure.
      ). A mutation in C3′H (ref
      • Chang M.C.Y.
      • Eachus R.A.
      • Trieu W.
      • Ro D.K.
      • Keasling J.D.
      Engineering Escherichia coli for production of functionalized terpenoids using plant P450s.
      ) in Arabidopsis that blocked formation of the G-unit, to produce only the H-unit, also induced severe dwarfism (
      • Franke R.
      • Hemm M.R.
      • Denault J.W.
      • Ruegger M.O.
      • Humphreys J.M.
      • Chapple C.
      Changes in secondary metabolism and deposition of an unusual lignin in the ref8 mutant of Arabidopsis.
      ), and a similar mutation in poplar caused aberrant accumulation of polyphenols (
      • Coleman H.D.
      • Park J.-Y.
      • Nair R.
      • Chapple C.
      • Mansfield S.D.
      RNAi-mediated suppression of p-coumaroyl-CoA 3-hydroxylase in hybrid poplar impacts lignin deposition and soluble secondary metabolism.
      ). However, secondary mutations in a component of an Arabidopsis transcriptional repressor complex, called Mediator5, rescued both growth and accumulation of H-lignin in the ref
      • Chang M.C.Y.
      • Eachus R.A.
      • Trieu W.
      • Ro D.K.
      • Keasling J.D.
      Engineering Escherichia coli for production of functionalized terpenoids using plant P450s.
      mutant background (
      • Bonawitz N.D.
      • Kim J.I.
      • Tobimatsu Y.
      • Ciesielski P.N.
      • Anderson N.A.
      • Ximenes E.
      • Maeda J.
      • Ralph J.
      • Donohoe B.S.
      • Ladisch M.
      • Chapple C.
      Disruption of Mediator rescues the stunted growth of a lignin-deficient Arabidopsis mutant.
      ). These results showed further that plants were not compromised by the new lignin monomer itself but by metabolites derived from the phenylpropanoid biosynthetic pathway that repressed expression of genes involved in growth. By overcoming such natural regulatory constraints, it might be possible to increase the range of valuable aromatic substances that can be incorporated into cell walls (
      • Muro-Villanueva F.
      • Mao X.
      • Chapple C.
      Linking phenylpropanoid metabolism, lignin deposition, and plant growth inhibition.
      ).

      Recalcitrance is a multiscale, multifaceted property

      Despite the genetic modification of lignin composition and degree of polymerization to improve enzymatic digestibility, various physical and chemical pretreatments, such as dilute acids (
      • Li C.
      • Knierim B.
      • Manisseri C.
      • Arora R.
      • Scheller H.V.
      • Auer M.
      • Vogel K.P.
      • Simmons B.A.
      • Singh S.
      Comparison of dilute acid and ionic liquid pretreatment of switchgrass: biomass recalcitrance, delignification and enzymatic saccharification.
      ), ammonia fiber expansion (
      • Teymouri F.
      • Laureano-Perez L.
      • Alizadeh H.
      • Dale B.E.
      Optimization of the ammonia fiber explosion (AFEX) treatment parameters for enzymatic hydrolysis of corn stover.
      ), and steam-expansion in water (
      • Mosier N.
      • Wyman C.
      • Dale B.E.
      • Elander R.
      • Lee Y.Y.
      • Holtzapple M.
      • Ladisch M.
      Features of promising technologies for pretreatment of lignocellulosic biomass.
      ), were needed before improvements from lignin modification could be observed. During steam-expansion, lignin is melted and redistributed into small globules, regardless of its composition (
      • Donohoe B.S.
      • Decker S.R.
      • Tucker M.P.
      • Himmel M.E.
      • Vinzant T.B.
      Visualizing lignin coalescence and migration through maize cell walls following thermochemical pretreatment.
      ). As lignin is thought to surround and coat microfibrils (Fig. 1C), it might be expected that melting would expose cellulose to cellulase activity. However, as steam-expanded biomass remained partially resistant to cellulase digestion, recalcitrance is more complicated than simply the occlusion of cellulose microfibrils at atomic scale. In poplar, negative correlations of sugar release and lignin content were observed only before pretreatment or in variants with low S-lignin (
      • Studer M.H.
      • DeMartini J.D.
      • Davis M.F.
      • Sykes R.W.
      • Davison B.
      • Keller M.
      • Tuskan G.A.
      • Wyman C.E.
      Lignin content in natural Populus variants affects sugar release.
      ). Some poplar variants with average lignin content exhibited exceptionally high saccharification. In a maize recombinant-inbred population, lignin abundance and saccharification yield were determined to be independent traits that were controlled by different genes (
      • Penning B.W.
      • Sykes R.W.
      • Babcock N.C.
      • Dugard C.K.
      • Held M.A.
      • Klimek J.F.
      • Shreve J.
      • Fowler M.
      • Ziebell A.
      • Davis M.F.
      • Decker S.R.
      • Turner G.B.
      • Mosier N.S.
      • Springer N.M.
      • Thimmapuram J.
      • et al.
      Genetic determinants for enzymatic digestion of lignocellulosic biomass are independent of those for lignin abundance in a maize recombinant inbred population.
      ). We proposed a new definition of recalcitrance, as those features of biomass that proportionately increase energy requirements in conversion processes, increase the cost and complexity of operations in the biorefinery, and/or reduce the recovery of biomass carbon into desired products (
      • McCann M.C.
      • Carpita N.C.
      Biomass recalcitrance: a multi-scale, multi-factor and conversion-specific property.
      ). The complexity of interactions that occur between cell-wall constituents, giving rise to recalcitrance, spans over 4 orders of magnitude: 1) molecular scale interactions among structurally complex polymers; 2) nanoscale assembly of individual and bundled cellulose microfibrils with hemicellulose and lignin; 3) mesoscale with wall domains of distinct composition within and between cells within a tissue; and 4) macroscale organization of cells and tissues that determine the chemo-physical properties of biomass (Fig. 3).
      Figure thumbnail gr3
      Figure 3Schematic figure illustrating length scales of plant biomass, its component polymers, and useful molecules and materials derived from them. Examples of poplar and sorghum (far left) at macroscale have mesoscale features of dense cellular organization in tissues (middle left micrograph, adapted from Ref.
      • Ciesielski P.N.
      • Wagner R.
      • Bharadwaj V.S.
      • Killgore J.
      • Mittal A.
      • Beckham G.T.
      • Decker S.R.
      • Himmel M.E.
      • Crowley M.F.
      Nanomechanics of cellulose deformation reveal molecular defects that facilitate natural deconstruction.
      ) and features of lignified secondary walls and compound middle lamellae (middle right micrograph, adapted from Ref.
      • Donaldson L.A.
      Lignification and lignin topochemistry—an ultrastructural view.
      ). Nanoscale structures comprise the cross-bridging of cellulose microfibrils by hemicelluloses in a co-extensive lignin matrix (adapted from Ref.
      • Ciesielski P.N.
      • Wagner R.
      • Bharadwaj V.S.
      • Killgore J.
      • Mittal A.
      • Beckham G.T.
      • Decker S.R.
      • Himmel M.E.
      • Crowley M.F.
      Nanomechanics of cellulose deformation reveal molecular defects that facilitate natural deconstruction.
      ) and cellulose microfibrils. At the molecular scale are several types of hemicelluloses and lignin with representative variations in linkage structure. Polymeric materials isolated from cell walls include cellulose nanocrystals and nanofibers (micrographs; courtesy of P. Ciesielski, National Renewable Energy Laboratory) and hemicelluloses that can then be functionalized with various chemical groups. These can be annealed to cellulose to form functionalized nanocrystals or nanofibers. Glucose and other monosaccharides from cellulose and hemicelluloses, and monolignols from depolymerization of polysaccharides and lignin can be catalytically transformed to a range of intermediates and subsequently deoxygenated using hydrogen to produce monomers for plastics, liquid fuel hydrocarbons and high-value aromatics, flavor compounds, and essential oils. Monomers for plastics include PLA, PHB, polybutylene-succinate (PBS), PET, and polyhydroxyurethane (PHU). Liquid hydrocarbon fuel substrates include paraffins, aromatics, levulinate, furfural, and hydroxymethylfurfural (HMF).

      The lignin-first biorefinery recovers both aromatics and sugars

      In the second-generation biorefinery concept, lignin was regarded as a waste product, only suitable for generating electricity by its combustion. However, as a consequence of new deconstruction technologies that preserve aromatic ring structure (
      • Bozell J.J.
      • Black S.K.
      • Myers M.
      • Cahill D.
      • Miller W.P.
      • Park S.
      Solvent fractionation of renewable woody feedstocks: Organosolv generation of biorefinery process streams for the production of biobased chemicals.
      ,
      • Labbé N.
      • Kline L.M.
      • Moens L.
      • Kim K.
      • Kim P.C.
      • Hayes D.G.
      Activation of lignocellulosic biomass by ionic liquid for biorefinery fractionation.
      ,
      • Parsell T.H.
      • Owen B.C.
      • Klein I.
      • Jarrell T.M.
      • Marcum C.L.
      • Haupert L.J.
      • Amundson L.M.
      • Kenttämaa H.I.
      • Ribeiro F.
      • Miller J.T.
      • Abu-Omar M.M.
      Cleavage and hydrodeoxygenation (HDO) of C–O bonds relevant to lignin conversion using Pd/Zn synergistic catalysis.
      ,
      • Socha A.M.
      • Parthasarathi P.
      • Shi J.
      • Pattathil S.
      • Whyte D.
      • Bergeron M.
      • George A.
      • Tran K.
      • Stavila V.
      • Venkatachalam S.
      • Hahn M.G.
      • Simmons B.A.
      • Singh S.
      Efficient biomass pretreatment using ionic liquids derived from lignin and hemicellulose.
      ), lignin is now a source of valuable co-products and is not a major factor of recalcitrance. Chemical catalytic systems, such as bimetallic Pd-Zn/C (
      • Parsell T.H.
      • Owen B.C.
      • Klein I.
      • Jarrell T.M.
      • Marcum C.L.
      • Haupert L.J.
      • Amundson L.M.
      • Kenttämaa H.I.
      • Ribeiro F.
      • Miller J.T.
      • Abu-Omar M.M.
      Cleavage and hydrodeoxygenation (HDO) of C–O bonds relevant to lignin conversion using Pd/Zn synergistic catalysis.
      ) or Ni/C (
      • Luo H.
      • Klein I.M.
      • Jiang Y.
      • Zhu H.
      • Liu B.
      • Kenttämaa H.I.
      • Abu-Omar M.M.
      Total utilization of Miscanthus biomass, lignin and carbohydrates, using earth abundant nickel catalyst.
      ), depolymerized all lignin from intact wood particles via β-O-4 bond cleavage, yielding about one-half of the product mixture as dimethoxypropylphenol and dihydroeugenol, derived from S- and G-lignins, respectively (
      • Parsell T.
      • Yohe S.
      • Degenstein J.
      • Jarrell T.
      • Klein I.
      • Gencer E.
      • Hewetson B.
      • Hurt M.
      • Kim J.I.
      • Choudhari H.
      • Saha B.
      • Meilan R.
      • Mosier N.
      • Ribeiro F.
      • Delgass W.N.
      • et al.
      A synergistic biorefinery based on catalytic conversion of lignin prior to cellulose starting from lignocellulosic biomass.
      ). The catalytic depolymerization of lignin (CDL) occurred without decomposition of cellulose or xylan, enabling the concept of the “lignin-first” biorefinery, where aromatic fuel substrates are removed before cellulose and other carbohydrates are processed (
      • Yang H.
      • Zhang X.
      • Luo H.
      • Liu B.
      • Shiga T.M.
      • Li X.
      • Kim J.I.
      • Rubinelli P.
      • Overton J.C.
      • Subramanyam V.
      • Abu-Omar M.
      • Chapple C.
      • Donohoe B.S.
      • Cooper B.R.
      • Mo H.
      • et al.
      Overcoming cellulose recalcitrance in woody biomass for the lignin-first biorefinery.
      ,
      • Ragauskas A.J.
      • Beckham G.T.
      • Biddy M.J.
      • Chandra R.
      • Chen F.
      • Davis M.F.
      • Davison B.H.
      • Dixon R.A.
      • Gilna P.
      • Keller M.
      • Langan P.
      • Naskar A.K.
      • Saddler J.N.
      • Tschaplinski T.J.
      • Tuskan G.A.
      • et al.
      Lignin valorization: improving lignin processing in the biorefinery.
      ,
      • Schutyser W.
      • Van den Bosch S.
      • Renders T.
      • De Boe T.
      • Koelewijn S.-F.
      • Dewaele A.
      • Ennaert T.
      • Verkinderen O.
      • Goderis B.
      • Courtin C.M.
      • Sels B.F.
      Influence of bio-based solvents on the catalytic reductive fractionation of birch wood.
      ,
      • Key R.E.
      • Bozell J.J.
      Progress toward lignin valorization via selective catalytic technologies and the tailoring of biosynthetic pathways.
      ).
      Third-generation drop-in fuels, fully compatible with existing engines and transportation infrastructure, include liquid hydrocarbons produced by chemical or enzymatic catalytic conversion of biomass-derived sugars and aromatics (
      • Huber G.W.
      • Shabaker J.W.
      • Dumesic J.A.
      Raney Ni–Sn catalyst for H production from biomass-derived hydrocarbons.
      ,
      • Wang T.
      • Nolte M.W.
      • Shanks B.H.
      Catalytic dehydration of C6 carbohydrates for the production of hydroxymethylfurfural (HMF) as a versatile platform chemical.
      ). The two main reaction products of CDL, dihydroeugenol and dimethoxypropylphenol, have been deoxygenated to high-octane molecules, propylbenzene and propylcyclohexane, using a selective Pt/Mo catalyst on multiwalled carbon nanotubes with >98% C9 hydrocarbon yield (
      • Yohe S.L.
      • Choudhari H.J.
      • Mehta D.D.
      • Dietrich P.J.
      • Detwiler M.D.
      • Akatay C.M.
      • Stach E.A.
      • Miller J.T.
      • Delgass W.N.
      • Agrawal R.
      • Ribeiro F.
      High-pressure vapor-phase hydrodeoxygenation of lignin-derived oxygenates to hydrocarbons by a PtMo bimetallic catalyst: product selectivity, reaction pathway, and structural characterization.
      ). Pathways that employ lignin as a substrate to replace commodity chemicals have also been envisioned (
      • Wellisch M.
      • Jungmeier G.
      • Karbowski A.
      • Patel M.K.
      • Rogulska M.
      Biorefinery systems—potential contributors to sustainable innovation.
      ,
      • Zakzeski J.
      • Bruijnincx P.C.A.
      • Jongerius A.L.
      • Weckhuysen B.M.
      The catalytic valorization of lignin for the production of renewable chemicals.
      ). As lignin comprises up to 25% of biomass and 40% of its energy content, the concept of the lignin-first biorefinery is critical to the efficient recovery and use of all biomass carbon.

      Redesigning biomass for the lignin-first biorefinery

      Armed with the capabilities of CDL, synthesis of aromatic end products derived from lignin in genetically modified plants is already possible. Transgenic poplar trees that made either S- or G-lignins produced dimethoxypropylphenol or dihydroeugenol, respectively, when CDL-treated, compared with WT poplar from which both compounds were derived (
      • Parsell T.H.
      • Owen B.C.
      • Klein I.
      • Jarrell T.M.
      • Marcum C.L.
      • Haupert L.J.
      • Amundson L.M.
      • Kenttämaa H.I.
      • Ribeiro F.
      • Miller J.T.
      • Abu-Omar M.M.
      Cleavage and hydrodeoxygenation (HDO) of C–O bonds relevant to lignin conversion using Pd/Zn synergistic catalysis.
      ). Separation of these fragrance and flavor molecules from each other is resolved by isolating their substrates in two different organisms. To address the large energy costs associated with separations of desired products from complex mixtures, a new paradigm should combine in planta design of molecules with ex planta processing for the most efficient production of useful renewable feedstocks for fuels and chemicals.
      Optimization of biomass for end uses in the lignin-first biorefinery can target features of recalcitrance beyond lignin. Regardless of downstream conversion process, the mechanical reduction of intact biomass to particles, called comminution, is energy-intensive. The interfaces between xylem cells of wood are elaborated into a compound middle lamella containing pectins, cellulose, and hemicelluloses, in addition to lignin and other phenolic substances (
      • Donaldson L.A.
      Lignification and lignin topochemistry—an ultrastructural view.
      ). Expression of pectin-degrading enzymes in Arabidopsis increased saccharification yield (
      • Tomassetti S.
      • Pontiggia D.
      • Verrascina I.
      • Reca I.B.
      • Francocci F.
      • Salvi G.
      • Cervone F.
      • Ferrari S.
      Controlled expression of pectic enzymes in Arabidopsis thaliana enhances biomass conversion without adverse effects on growth.
      ). By contrast, enhanced pectin synthesis from overexpression of homogalacturonan synthase genes decreased saccharification yield in poplar (
      • Biswal A.K.
      • Atmodjo M.A.
      • Pattathil S.
      • Amos R.A.
      • Yang X.
      • Winkeler K.
      • Collins C.
      • Mohanty S.S.
      • Ryno D.
      • Tan L.
      • Gelineo-Albersheim I.
      • Hunt K.
      • Sykes R.W.
      • Turner G.B.
      • Ziebell A.
      • et al.
      Working towards recalcitrance mechanisms: increased xylan and homogalacturonan production by overexpression of GAlactUronosylTransferase12 (GAUT12) causes increased recalcitrance and decreased growth in Populus.
      ), whereas blocking synthesis increased yield (
      • Biswal A.K.
      • Atmodjo M.A.
      • Li M.
      • Baxter H.L.
      • Yoo C.G.
      • Pu Y.
      • Lee Y.C.
      • Mazarei M.
      • Black I.M.
      • Zhang J.-Y.
      • Ramanna H.
      • Bray A.L.
      • King Z.R.
      • LaFayette P.R.
      • Pattathil S.
      • et al.
      Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis.
      ). A relatively minor wall constituent called rhamnogalacturonan-I is also a determinant of cell-cell adhesion. Oxidation of lignin by sodium chlorite and extraction of hemicelluloses and pectins by alkali were both required to achieve fiber cell separation in wood particles from poplar (
      • Yang H.
      • Benatti M.R.
      • Karve R.A.
      • Fox A.
      • Meilan R.
      • Carpita N.C.
      • McCann M.C.
      Rhamnogalacturonan I is a determinant of cell-cell adhesion in poplar wood.
      ). Xylanase alone was ineffective in permitting cell separation of delignified materials, but the addition of a rhamnogalacturonan-I lyase caused dissolution of the compound middle lamella. When an Arabidopsis gene encoding a rhamnogalacturonan-I lyase was expressed in poplar, disruption of cell-cell adhesion, reduction in particle size upon grinding, and saccharification yields were all enhanced over WT controls without reducing biomass yield (
      • Yang H.
      • Benatti M.R.
      • Karve R.A.
      • Fox A.
      • Meilan R.
      • Carpita N.C.
      • McCann M.C.
      Rhamnogalacturonan I is a determinant of cell-cell adhesion in poplar wood.
      ). The transgenic poplar wood was more easily fragmented under mechanical stress, potentially reducing energy inputs for comminution.
      Maleic acid is a catalyst that depolymerizes xylans from Arabidopsis cell walls, regardless of lignin composition, and converts xylose to furfural in a two-step reaction (
      • Ciesielski P.N.
      • Resch M.G.
      • Hewetson B.
      • Killgore J.P.
      • Curtin A.
      • Anderson N.
      • Chiaramonti A.N.
      • Hurley D.C.
      • Sanders A.
      • Himmel M.E.
      • Chapple C.
      • Mosier N.
      • Donohoe B.S.
      Engineering plant cell walls: tuning lignin monomer composition for deconstructable biofuel feedstocks or resilient biomaterials.
      ). However, Arabidopsis plants with high S-lignin content showed disruption of cell-cell adhesion between fiber cells upon treatment with maleic acid (
      • Ciesielski P.N.
      • Resch M.G.
      • Hewetson B.
      • Killgore J.P.
      • Curtin A.
      • Anderson N.
      • Chiaramonti A.N.
      • Hurley D.C.
      • Sanders A.
      • Himmel M.E.
      • Chapple C.
      • Mosier N.
      • Donohoe B.S.
      Engineering plant cell walls: tuning lignin monomer composition for deconstructable biofuel feedstocks or resilient biomaterials.
      ). In poplar, CDL treatment results in greatly enhanced saccharification yields in high-S-lignin genetic variants, perhaps as a result of increased fiber cell separation and, thus, access of cellulases or chemical catalysts. Significant recalcitrance to enzymatic digestion and maleic acid–catalyzed conversion to fuel molecules remained because of cellulose crystallinity (Fig. 4) (
      • Yang H.
      • Zhang X.
      • Luo H.
      • Liu B.
      • Shiga T.M.
      • Li X.
      • Kim J.I.
      • Rubinelli P.
      • Overton J.C.
      • Subramanyam V.
      • Abu-Omar M.
      • Chapple C.
      • Donohoe B.S.
      • Cooper B.R.
      • Mo H.
      • et al.
      Overcoming cellulose recalcitrance in woody biomass for the lignin-first biorefinery.
      ). Impregnating ferrous iron into biomass, as a co-catalyst with dilute acid, caused the separation of dense aggregates of cellulose microfibrils (
      • Wei H.
      • Donohoe B.S.
      • Vinzant T.B.
      • Ciesielski P.N.
      • Wang W.
      • Gedvilas L.M.
      • Zeng Y.
      • Johnson D.K.
      • Ding S.-Y.
      • Himmel M.E.
      • Tucker M.P.
      Elucidating the role of ferrous ion cocatalyst in enhancing dilute acid pretreatment of lignocellulosic biomass.
      ). Expression of a fusion protein, comprising several iron-binding peptides with a carbohydrate-binding module, secreted into rice and Arabidopsis cell walls, showed significant increases in iron accumulation and saccharification yield compared with WT (
      • Yang H.
      • Wei H.
      • Ma G.
      • Antunes M.S.
      • Vogt S.
      • Cox J.
      • Zhang X.
      • Liu X.
      • Bu L.
      • Gleber S.C.
      • Carpita N.C.
      • Makowski L.
      • Himmel M.E.
      • Tucker M.P.
      • McCann M.C.
      • et al.
      Cell wall targeted in planta iron accumulation enhances biomass conversion and seed iron concentration in Arabidopsis and rice.
      ). Site-selective delivery of ferritin catalysts in transgenic lines of Arabidopsis yielded 12–18% more shoot biomass and released 20–30% more sugar following pretreatment, relative to control plants without iron incorporation (
      • Wei H.
      • Yang H.
      • Ciesielski P.N.
      • Donohoe B.S.
      • McCann M.C.
      • Murphy A.S.
      • Peer W.A.
      • Ding S.-Y.
      • Himmel M.E.
      • Tucker M.P.
      Transgenic ferritin overproduction enhances thermochemical pretreatments in Arabidopsis.
      ,
      • Lin C.-Y.
      • Jakes J.E.
      • Donohoe B.S.
      • Ciesielski P.N.
      • Yang H.
      • Gleber C.S.
      • Vogt S.
      • Ding S.-Y.
      • Peer W.A.
      • Murphy A.S.
      • McCann M.C.
      • Himmel M.E.
      • Tucker M.P.
      • Wei H.
      Directed plant cell wall accumulation of iron: embedding co-catalyst for efficient biomass conversion.
      ). Delivery of metal catalysts throughout the cell-wall structure and creating functionalized sites ready for catalytic transformations increased the effective surface area for catalysis.
      Figure thumbnail gr4
      Figure 4Rates of enzymatic hydrolysis of milled wood particles from WT and transgenic poplar lines from materials treated with TFA before or after CDL. Enzymatic hydrolysis was initiated by the addition of Cellic® Ctec2 (Novozymes) to 5 mg of milled particles, followed by incubation at 37 °C for up to 72 h. Digestion of untreated particles (black lines) is not improved compared with digestion of those incubated in TFA at –20 °C (blue lines). CDL-treated particles (green lines) show greatly increased digestibility for all lines. Even when lignin was removed by CDL, the high-S-lignin transgenic lines showed higher initial rates of digestion. All materials readily swell in TFA at –20 °C after CDL treatment, and the resulting improvement in digestibility illustrates the recalcitrance of cellulose crystallinity (red lines). WT poplar hybrids (Populus tremula × P. alba) were converted to high-S-lignin lines (High S) by overexpression of F5H or to high-G-lignin lines (High G) by RNAi knockdown of F5H; high-OH-G-lignin lines (High OH-G) were generated by RNAi knockdown of COMT. Data adapted from Ref.
      • Yang H.
      • Zhang X.
      • Luo H.
      • Liu B.
      • Shiga T.M.
      • Li X.
      • Kim J.I.
      • Rubinelli P.
      • Overton J.C.
      • Subramanyam V.
      • Abu-Omar M.
      • Chapple C.
      • Donohoe B.S.
      • Cooper B.R.
      • Mo H.
      • et al.
      Overcoming cellulose recalcitrance in woody biomass for the lignin-first biorefinery.
      .

      Deconstruction of cellulosic residues is facilitated by lignin removal

      Lignin deconstruction leads to improved feedstock for subsequent catalytic conversion of the carbohydrate-enriched residues. CDL treatment doubled the rates of enzymatic hydrolysis of cellulosic residues of poplar genetic variants (
      • Yang H.
      • Zhang X.
      • Luo H.
      • Liu B.
      • Shiga T.M.
      • Li X.
      • Kim J.I.
      • Rubinelli P.
      • Overton J.C.
      • Subramanyam V.
      • Abu-Omar M.
      • Chapple C.
      • Donohoe B.S.
      • Cooper B.R.
      • Mo H.
      • et al.
      Overcoming cellulose recalcitrance in woody biomass for the lignin-first biorefinery.
      ). Higher initial rates of saccharification were observed in the high-S-lignin lines although all of the lignin had been removed (Fig. 4). Thus, lignin composition “imprinted” its recalcitrance on cellulosic structure even after catalytic removal.
      Cellulose solvents, such as N-methylmorpholine N-oxide (
      • Fink H.P.
      • Weigel P.
      • Purz H.J.
      • Ganster J.
      Structure formation of regenerated cellulose materials from NMMO-solutions.
      ), or ionic liquids, such as 1-butyl-3-methylimidazolium chloride (
      • Socha A.M.
      • Parthasarathi P.
      • Shi J.
      • Pattathil S.
      • Whyte D.
      • Bergeron M.
      • George A.
      • Tran K.
      • Stavila V.
      • Venkatachalam S.
      • Hahn M.G.
      • Simmons B.A.
      • Singh S.
      Efficient biomass pretreatment using ionic liquids derived from lignin and hemicellulose.
      ,
      • Pinkert A.
      • Marsh K.N.
      • Pang S.S.
      • Staiger M.P.
      Ionic liquids and their interaction with cellulose.
      ), can solubilize cellulose without inducing extensive decomposition. Cellulose was swollen and dissolved at low temperature in TFA with minimal decomposition, and this treatment greatly enhanced saccharification yield or catalytic conversion to substrates for the liquid hydrocarbon fuels, levulinic acid and hydroxymethylfurfural (
      • Shiga T.M.
      • Xiao W.
      • Yang H.
      • Zhang X.
      • Olek A.T.
      • Donohoe B.S.
      • Makowski L.
      • Hou T.
      • Zhang Y.
      • Zhao T.
      • McCann M.C.
      • Carpita N.C.
      • Mosier N.S.
      Enhanced rates of enzymatic saccharification and catalytic synthesis of biofuel substrates in gelatinized cellulose generated by trifluoroacetic acid.
      ). Cellulose swelling was blocked to a large extent by lignin in poplar wood particles, but CDL treatment eliminated this restriction (
      • Yang H.
      • Zhang X.
      • Luo H.
      • Liu B.
      • Shiga T.M.
      • Li X.
      • Kim J.I.
      • Rubinelli P.
      • Overton J.C.
      • Subramanyam V.
      • Abu-Omar M.
      • Chapple C.
      • Donohoe B.S.
      • Cooper B.R.
      • Mo H.
      • et al.
      Overcoming cellulose recalcitrance in woody biomass for the lignin-first biorefinery.
      ). The amorphous forms of cellulose generated by low-temperature swelling of delignified poplar approached complete digestion in 6 h, with the highest initial rates in the high-S-lignin materials, compared with only 60% digestion after 72 h in control digests (Fig. 4), and at least 10-fold increases in yields of maleic acid–catalyzed production of levulinic acid and hydroxymethylfurfural (
      • Yang H.
      • Zhang X.
      • Luo H.
      • Liu B.
      • Shiga T.M.
      • Li X.
      • Kim J.I.
      • Rubinelli P.
      • Overton J.C.
      • Subramanyam V.
      • Abu-Omar M.
      • Chapple C.
      • Donohoe B.S.
      • Cooper B.R.
      • Mo H.
      • et al.
      Overcoming cellulose recalcitrance in woody biomass for the lignin-first biorefinery.
      ). Because of its low boiling point, facile recycling of TFA in a closed system and a continuous-flow cellulose solubilization are possible (
      • Shiga T.M.
      • Xiao W.
      • Yang H.
      • Zhang X.
      • Olek A.T.
      • Donohoe B.S.
      • Makowski L.
      • Hou T.
      • Zhang Y.
      • Zhao T.
      • McCann M.C.
      • Carpita N.C.
      • Mosier N.S.
      Enhanced rates of enzymatic saccharification and catalytic synthesis of biofuel substrates in gelatinized cellulose generated by trifluoroacetic acid.
      ).

      Utilizing sugars and aromatics for higher-value co-products

      Biofuel producers could benefit from diverse product portfolios that provide alternative revenue sources to ride out market fluctuations in oil price. The success of the petroleum industry is based on a small number of chemicals, including benzene, toluene, xylene, and styrene, that are oxygenated chemically to make substrates for plastics, synthetic textiles, and other materials (
      • Parsell T.
      • Yohe S.
      • Degenstein J.
      • Jarrell T.
      • Klein I.
      • Gencer E.
      • Hewetson B.
      • Hurt M.
      • Kim J.I.
      • Choudhari H.
      • Saha B.
      • Meilan R.
      • Mosier N.
      • Ribeiro F.
      • Delgass W.N.
      • et al.
      A synergistic biorefinery based on catalytic conversion of lignin prior to cellulose starting from lignocellulosic biomass.
      ,
      • Wang T.
      • Nolte M.W.
      • Shanks B.H.
      Catalytic dehydration of C6 carbohydrates for the production of hydroxymethylfurfural (HMF) as a versatile platform chemical.
      ). Whereas the petroleum industry must functionalize hydrocarbons with oxygen, biomass molecules are already highly oxygenated. Chemical or enzymatic reactions with biomass-derived substrates therefore need to be highly selective to preserve desired functional groups in target products. Tandem catalytic reactions, where two catalysts are in the same reaction vessel, and the product of the first reaction becomes the substrate for the second, have been demonstrated for depolymerization and subsequent deoxygenation of cellulose and xylan to alkanes, alkenes and furan-based fuels (
      • Huber G.W.
      • Shabaker J.W.
      • Dumesic J.A.
      Raney Ni–Sn catalyst for H production from biomass-derived hydrocarbons.
      ,
      • Wang T.
      • Nolte M.W.
      • Shanks B.H.
      Catalytic dehydration of C6 carbohydrates for the production of hydroxymethylfurfural (HMF) as a versatile platform chemical.
      ,
      • Wegenhart B.L.
      • Yang L.N.
      • Kwan S.C.
      • Harris R.
      • Kenttämaa H.I.
      • Abu-Omar M.M.
      From furfural to fuel: synthesis of furoins by organocatalysis and their hydrodeoxygenation by cascade catalysis.
      ). Controlled fractionation of biomass with consideration of downstream catalytic upgrading provides several value-added streams for xylans to furfural (
      • Vinueza N.R.
      • Kim E.S.
      • Gallardo V.A.
      • Mosier N.S.
      • Abu-Omar M.M.
      • Carpita N.C.
      • Kenttäma H.I.
      Tandem mass spectrometric characterization of the conversion of xylose to furfural.
      ), lignin to aromatics and dicarboxylic acids (
      • Zeng J.
      • Yoo C.G.
      • Wang F.
      • Pan X.
      • Vermerris W.
      • Tong Z.
      Biomimetic Fenton-catalyzed lignin depolymerization to high-value aromatics and dicarboxylic acids.
      ), and cellulose to hydroxymethylfurfural (
      • Hewetson B.
      • Zhang X.
      • Mosier N.S.
      Enhanced acid-catalyzed biomass conversion to hydroxymethylfurfural following cellulose solvent-and organic solvent-based lignocellulosic fractionation pretreatment.
      ).
      Thermoset plastics and polymers are materials that remain in a permanent solid state after being cured and include epoxy, silicone, and polyurethane. Polymers within the material cross-link during the curing process to form irreversible bonds, such that thermosets will not melt even when exposed to extremely high temperatures. Lignin-derived monomers have been incorporated into polymers to create new bio-based materials with improved performance characteristics compared with fossil fuel–derived thermoset materials (
      • Zhao S.
      • Abu-Omar M.M.
      Biobased epoxy nanocomposites derived from lignin-based monomers.
      ,
      • Chen C.-H.
      • Tung S.-H.
      • Jeng R.-J.
      • Abu-Omar M.A.
      • Lin C.-H.
      A facile strategy to achieve fully bio-based epoxy thermosets from eugenol.
      ) and also with carbohydrate-derived monomers (
      • Jiang Y.
      • Ding D.
      • Zhao S.
      • Zhu H.
      • Kenttämaa H.I.
      • Abu-Omar M.M.
      Renewable thermosets based on lignin and carbohydrate derived monomers.
      ). Chemical methods that improved molecular weight, orientation, or the number of functional groups on monomers increased the cross-linking density of bio-based epoxy networks based on the dihydroeugenol product from CDL (
      • Zhao S.
      • Abu-Omar M.M.
      Renewable epoxy networks derived from lignin-based monomers: effect of cross-linking density.
      ). Poplar fibers have also been directly incorporated into composites with polylactic acid (PLA) as a replacement for conventional carbon nanofibers that reinforce polymers for large-scale 3D printing applications (
      • Zhao X.
      • Tekinalp H.
      • Meng X.
      • Ker D.
      • Benson B.
      • Yunqiao P.
      • Ragauskas A.J.
      • Wang Y.
      • Li K.
      • Webb E.
      • Gardner D.J.
      • Anderson J.
      • Ozcan S.
      Poplar as biofiber reinforcement in composites for large-scale 3D printing.
      ).
      In contrast to thermosets, thermoplastics can be melted, and some of their monomers may be recycled. Early efforts to synthesize these materials from renewable resources focused on synthesis of PLA, polyterephthalate (PTT), and polybutylene-succinate by microbes from glucose-derived substrates, and polyhydroxyalkanoate (PHA) directly by microbes (
      • Reddy M.M.
      • Vivekanandhan S.
      • Misra M.
      • Bhatia S.K.
      • Mohanty A.K.
      Biobased plastics and bionanocomposites: Current status and future opportunities.
      ). More recently, bio-based polyethylene-terephthalate (PET) polymers, made from terephthalates and ethylene glycol, have been widely used to make plastic bottles (
      • Sheldon R.A.
      Green and sustainable manufacture of chemicals from biomass: State of the art.
      ). The entire pathway to polyhydroxybutyrate (PHB) was engineered in cotton almost 25 years ago (
      • John M.E.
      • Keller G.
      Metabolic pathway engineering in cotton: Biosynthesis of polyhydroxybutyrate in fiber cells.
      ) and more recently in the bioenergy crop switchgrass (
      • Somleva M.N.
      • Snell K.D.
      • Beaulieu J.J.
      • Peoples O.P.
      • Garrison B.R.
      • Patterson N.A.
      Production of polyhydroxybutyrate in switchgrass, a value-added co-product in an important lignocellulosic biomass crop.
      ). Routes for the biological synthesis of polyhydroxyurethane have been envisioned (
      • Nohra B.
      • Candy L.
      • Blanco J.-F.
      • Guerin C.
      • Raoul Y.
      • Mouloungui Z.
      From petrochemical polyurethanes to biobased polyhydroxyurethanes.
      ).
      Although bio-based plastics are now in commercial use, the key question is how to circularize the plastics economy. Insignificant degradation was observed for polyethylene or polypropylene plastics after almost 2 years of wet composting, even when combined in composites with cellulose or starch (
      • Gómez E.F.
      • Michel Jr., F.C.
      Biodegradability of conventional and bio-based plastics and natural fiber composites during composting, anaerobic digestion and long-term soil incubation.
      ). The PLAs and their composites were biodegradable, but PETs and PTTs were not (
      • Chen L.
      • Pelton R.E.O.
      • Smith T.M.
      Comparative life cycle assessment of fossil and bio-based polyethylene terephthalate (PET) bottles.
      ,
      • Emadian S.M.
      • Onay T.T.
      • Demirel B.
      Biodegradation of bioplastics in natural environments.
      ). Despite the reduction of CO2 emissions by synthesis of plant-based plastics, the lack of true biodegradability imposes a major disposal and environmental pollution cost (
      • Chen L.
      • Pelton R.E.O.
      • Smith T.M.
      Comparative life cycle assessment of fossil and bio-based polyethylene terephthalate (PET) bottles.
      ). By contrast, substantial disintegration of PHA-based plastics, such as PHB, was observed (
      • Gómez E.F.
      • Michel Jr., F.C.
      Biodegradability of conventional and bio-based plastics and natural fiber composites during composting, anaerobic digestion and long-term soil incubation.
      ). Cellulose, which is used primarily for fiber and production of films, can be derivatized with several types of esters engendering a broad range of “plastic-like” coatings and other fully biodegradable materials (
      • Reddy M.M.
      • Vivekanandhan S.
      • Misra M.
      • Bhatia S.K.
      • Mohanty A.K.
      Biobased plastics and bionanocomposites: Current status and future opportunities.
      ). Dissolution of cellulose in solvents such as TFA represents another route to green plastics (
      • Bayer I.S.
      • Guzman-Puyol S.
      • Heredia-Guerrero J.A.
      • Ceseracciu L.
      • Pignatelli F.
      • Ruffilli R.
      • Cingolani R.
      • Athanassiou A.
      Direct transformation of edible vegetable waste into bioplastics.
      ). In a circular economy, monomers should be recovered and reusable for subsequent polymerization without adversely affecting material quality. However, the combination of low market value and poor accumulation rates of plant-based plastics might represent an insurmountable challenge in the short run, favoring the production of higher-value coproducts from biomass (
      • Yang M.
      • Baral N.R.
      • Simmons B.A.
      • Mortimer J.C.
      • Shih P.M.
      • Scown C.D.
      Accumulation of high-value bioproducts in planta can improve the economics of advanced biofuels.
      ). In that regard, chemical modifications of lignins into high-value products, such as aromas, flavors, and essential oils, represents a potential economic incentive for biorefineries (
      • Parsell T.
      • Yohe S.
      • Degenstein J.
      • Jarrell T.
      • Klein I.
      • Gencer E.
      • Hewetson B.
      • Hurt M.
      • Kim J.I.
      • Choudhari H.
      • Saha B.
      • Meilan R.
      • Mosier N.
      • Ribeiro F.
      • Delgass W.N.
      • et al.
      A synergistic biorefinery based on catalytic conversion of lignin prior to cellulose starting from lignocellulosic biomass.
      ,
      • Laurichesse S.
      • Avérous L.
      Chemical modification of lignins: towards biobased polymers.
      ).

      Derivatized cellulose nanocrystals and nanofibers as novel materials

      Beyond biomass-derived sugars and aromatics, larger molecular structures can be isolated as products from cell walls. When pulped wood particles are treated with acids, crystalline particles of relatively uniform sizes are recovered, derived from breakage of crystalline cellulose microfibrils. The processing yields two types of materials, cellulose nanocrystals (CNCs) and cellulose nanofibers (CNFs), derivatives of which are used for several kinds of synthetic materials as replacements for plastics (Fig. 3) (
      • Moon R.J.
      • Martini A.
      • Nairn J.
      • Simonsen J.
      • Youngblood J.
      Cellulose nanomaterials review: structure, properties and nanocomposites.
      ,
      • Zhu H.
      • Luo W.
      • Ciesielski P.N.
      • Fang Z.
      • Zhu J.Y.
      • Henriksson G.
      • Himmel M.E.
      • Hu L.
      Wood-derived materials for green electronics, biological devices, and energy applications.
      ). Equivalent glucan chain lengths and cellulose microfibril dimensions are likely to be found in lignocellulosic biomass, but microfibrils are degraded to a certain extent during pulping and acid pretreatments (
      • Snyder J.L.
      • Timell T.E.
      The degree of polymerization of the cellulose component of balsam fir.
      ). Microfibrils from ramie plants showed, by small-angle neutron scattering, a periodicity between ordered and disordered domains of only 200–300 residues (
      • Nishiyama Y.
      • Kim U.-J.
      • Kim D.-Y.
      • Katsumata K.S.
      • May R.P.
      • Langan P.
      Periodic disorder along ramie cellulose microfibrils.
      ,
      • Nickerson R.F.
      • Habrle J.A.
      Cellulose intercrystalline structure.
      ,
      • Nelson M.L.
      • Tripp V.W.
      Determination of the leveling-off degree of polymerization of cotton and rayon.
      ). Three-dimensional electron tomography of cellulose in maize stover revealed a nanoscale curvature that may occur from the predicted twist in the microfibrils due to the bond angle formed between glucose residues of the (1→4)-β-d-glucan chains (
      • Ciesielski P.N.
      • Matthews J.F.
      • Tucker M.P.
      • Beckham G.T.
      • Crowley M.F.
      • Himmel M.E.
      • Donohoe B.S.
      3D electron tomography of pretreated biomass informs atomic modeling of cellulose microfibrils.
      ). Nanomanipulation by atomic force microscopy, imaging by transmission EM, and dynamic modeling revealed kink defects resulting from the bending stress at the 200–300-residue-length scale (
      • Ciesielski P.N.
      • Wagner R.
      • Bharadwaj V.S.
      • Killgore J.
      • Mittal A.
      • Beckham G.T.
      • Decker S.R.
      • Himmel M.E.
      • Crowley M.F.
      Nanomechanics of cellulose deformation reveal molecular defects that facilitate natural deconstruction.
      ), which might correspond to the disordered domains that break to form CNCs. Advantages of mechanical strength, flexibility, and nanostructuration make nanocellulose suitable for optoelectronics, wearable electronics, antibacterial coatings, packaging, mechanically reinforced polymer composites, tissue scaffolds, drug delivery, biosensors, energy storage, catalysis, environmental remediation, and electrochemically controlled separation technologies (
      • Zhu H.
      • Luo W.
      • Ciesielski P.N.
      • Fang Z.
      • Zhu J.Y.
      • Henriksson G.
      • Himmel M.E.
      • Hu L.
      Wood-derived materials for green electronics, biological devices, and energy applications.
      ,
      • Thomas B.
      • Raj M.C.
      • Athira K.B.
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      • Joy J.
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      ).
      Potential exists to modify, using chemical catalysis or synthetic biology, molecules that hydrogen-bond or sterically bond to cellulose. One of the more established derivatizations of CNCs and CNFs is through 2,2,6,6-tetramethylpiperidine 1-oxyl radical–catalyzed oxidation of the surface residues to glucuronosyl units (
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      ), giving a surface charge to the particles and fibers, to which esters with a range of functional groups can be attached. Xyloglucan oligomers have been used as acceptors to splice onto the reducing ends of xyloglucan polymers, which then hydrogen-bond to cellulose microfibrils, coating the surfaces. This technology uses xyloglucan endo-β-transglucosylase, an enzyme that cuts and rejoins hemicellulosic xyloglucans (
      • Fry S.C.
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      Xyloglucan endotransglycosylase, a new wall-loosening enzyme-activity from plants.
      ,
      • Nishitani K.
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      Endoxyloglucan transferase, a novel class of glycosyltransferase that catalyzes transfer of a segment of xyloglucan molecule to another xyloglucan molecule.
      ), where the reducing end of the acceptor can be derivatized in a variety of ways to add fluorescence markers, electrophiles, amines, or thiol groups to cellulose (Fig. 3) (
      • Teeri T.T.
      • Brumer III, H.
      • Daniel G.
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      Biomimetic engineering of cellulose-based materials.
      ). These groups change the biochemical or biophysical characteristics of the cellulose. For example, derivatizing cellulose with surface polymers improves performance of cellulose biocomposites as packaging materials (
      • Teeri T.T.
      • Brumer III, H.
      • Daniel G.
      • Gatenholm P.
      Biomimetic engineering of cellulose-based materials.
      ). The CNFs contribute strength to the biocomposite, whereas the polymer sheath improves its interfacial stability (
      • Teeri T.T.
      • Brumer III, H.
      • Daniel G.
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      Biomimetic engineering of cellulose-based materials.
      ). In addition to fluorescence tagging of monolignols (
      • Pandey J.L.
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      • Diehl B.G.
      • Richard T.L.
      • Chen G.
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      A versatile click-compatible monolignol probe to study lignin deposition in plant cell walls.
      ,
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      ), use of a suite of biocompatible small molecule reactions, called “click chemistry,” has been employed to generate fluorescence probes for cell-wall polysaccharide synthesis and dynamics in vivo (
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      Small molecule probes for plant cell wall polysaccharide imaging.
      ). For example, an alkynylated fluorescent fucose derivative was incorporated into rhamnogalacturonan-I via a nucleotide-sugar salvage pathway and subsequently detected in the cell walls of living root cells (
      • Anderson C.T.
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      Metabolic click-labeling with a fucose analog reveals pectin delivery, architecture, and dynamics in Arabidopsis cell walls.
      ). Further surface modifications can be made by attaching propargyl-amino groups through reductive amination to generate reactive groups suitable for these applications (
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      ). Xyloglucan modifications have been extended to functionalize CNCs (
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      Self-organization of cellulose nanocrystals adsorbed with xyloglucan oligosaccharide-poly(ethyleneglycol)-polystyrene triblock copolymer.
      ); CNFs have been modified to attach xyloglucan-RGD peptides to enhance cell adhesion and proliferation of human endothelial cells in tissue repair (
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      ).
      Two key problems with using xyloglucan endo-β-transglucosylase to attach functionalized oligomers to xyloglucan are that only a single functional group is added per polysaccharide and that xyloglucan is a primary wall polysaccharide in low abundance in secondary wall–rich biomass. The genes encoding the synthesis of (fucogalacto)xyloglucan have been identified (
      • Scheller H.V.
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      Hemicelluloses.
      ), as have those involved in synthesis and regulation of many other hemicellulosic polysaccharides of secondary walls (
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      ,
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      ). Thus, ectopic expression of the xyloglucan synthesis machinery in fiber cells during secondary wall development could be one strategy to increase xyloglucan amounts. However, an easier route to functionalize not only xyloglucan, but many other polysaccharides, is the expression of a galactose oxidase, which converts the O-6 residues to reactive aldehydes (
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      ). Oxidation of galactosyl residues of tamarind seed xyloglucan or guar galactomannan and attachment to CNFs made hydro- and aerogels of variable elastic and compression moduli and porosity (
      • Ghafar A.
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      ), as well as composite films of variable strength with CNFs and galactose-oxidized xyloglucans and guar gums (
      • Lucenius J.
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      Nanocomposite films based on cellulose nanofibrils and water-soluble polysaccharides.
      ). The significance of these findings lies also in differences in secondary wall composition of different species, considering the (galactogluco)mannan-rich gymnosperm wood, (arabino)xylan-rich angiosperm wood, and phenylpropanoid– and hydroxycinnamic acid–rich grasses (
      • Sarkar P.
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      • Auer M.
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      ), all of which have polysaccharides with varying degrees of galactosylation that can be modified. It is conceivable that galactose oxidase could be expressed ectopically during the late stages of secondary wall formation to functionalize hemicelluloses in situ that serve as attachment sites for products produced by synthetic pathways. High-value products could be synthesized, secreted, and stored in cell walls until recovered from biomass by chemical cleavage.

      Future directions

      Plants invest considerable thermodynamic potential in generating complex polymers, and the chemical moieties in these structures hold tremendous value as useful building blocks for chemical co-products. To produce hydrocarbon fuels from cell-wall polysaccharides and lignin, deoxygenation reactions must proceed to full chemical reduction, but for chemical products, reactions must necessarily be highly selective to preserve desirable functional chemical groups.
      A major consideration in the design of synthetic pathways is to minimize carbon losses in reaction steps from intact biomass to target products. Bio-based terephthalic acid synthesis demonstrates the need to consider an overall hybrid conversion pathway when determining atom efficiency. In one route, malic acid synthesized fermentatively can be dimerized and reacted via Diels–Alder chemistry to terephthalic acid (
      • Kraus G.A.
      • Riley S.
      • Cordes T.
      Aromatics from pyrones: para-Substituted alkyl benzoates from alkenes, coumalic acid and methyl coumalate.
      ,
      • Lee J.J.
      • Kraus G.A.
      Divergent Diels-Alder methodology from methyl coumalate toward functionalized aromatics.
      ), whereas muconic acid can be produced fermentatively and then isomerized and reacted via a Diels–Alder reaction to the diacid (
      • Suastegui M.
      • Matthiesen J.E.
      • Carraher J.M.
      • Hernandez N.
      • Rodriguez Quiroz N.
      • Okerlund A.
      • Cochran E.W.
      • Shao Z.
      • Tessonnier J.-P.
      Combining metabolic engineering and electrocatalysis: application to the production of polyamides from sugar.
      ). Of these two pathways, the route through muconic acid has the higher theoretical atom efficiency, but malic acid by itself has higher carbon efficiency than does muconic acid. Setting an overarching aim of discovering atom- and energy-efficient pathways allows a larger exploration space that encompasses plant, microbial, and chemical catalysts to achieve an optimized solution.
      The design of hybrid catalytic transformations requires synthesis and design of abundant and inexpensive catalyst materials, such as iron and nickel, that are tolerant of biogenic impurities, detailed kinetic and mechanistic measurements, and modeling of catalyst structures and reaction pathways. In turn, knowledge from hybrid catalyst systems could inform selection of microbial strain and fermentation media and suggest additional genetic modifications of plants. Integration of chemical and biochemical catalyses is needed to exploit synergies that reduce energy inputs and maximize atom economy in the biorefinery.
      Efficient production of target compounds in plants will require a systems-level understanding of metabolism and constraints, including tradeoffs between carbon fluxes and cellular energy balances. Multiple interconnected levels of gene regulation, genetic redundancy involving large gene families, compartmentation of metabolic activities in different organelles, and multicellularity together increase metabolic complexity in plants, making the design-build-test-improve engineering cycle more challenging than in that for microbial systems. Future technologies will facilitate both plant metabolic engineering itself and implementation strategies to engineer crops or plant cell cultures as bioproduction systems.
      Cross-links among plant cell-wall biopolymers generate nanoscale architectures and distinct mesoscale domains that have dramatically different properties than those observed in mixtures of biopolymers. The disparity between theoretical and actual yields of liquid hydrocarbons and high-value chemicals using various chemical catalytic systems is a consequence of this structural complexity. Understanding how genes control higher-order architectural features of biomass, and how those features then impact performance in conversion processes, could facilitate catalyst design and reduce reactor residence times and reduce energy inputs for the mechanical reduction of biomass to particles. However, this structural complexity can also be leveraged in the design of biorefinery operations, taking advantage of higher-value products that make the economics work for the large-volume, low-margin production of fuel. The examples that we discuss here represent a small fraction of potential biomass-derived products. The creativity of plant biologists is needed to test the constraints of how we co-opt nature to build cell walls that fulfill their functions in the living plant while providing all of the material comforts of modern life.

      Acknowledgments

      We thank our colleagues in the Center for Direct Catalytic Conversion of Biomass to Biofuels for a decade of insights, interdisciplinary engagement, and friendship.

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