Redesigning plant cell walls for the biomass-based bioeconomy

Abstract

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 (1). 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 (2), 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 (3). Some natural plant products have nutritional or pharmaceutical value and form the basis of foods, supplements, and drugs (4–6). Others govern interactions of the plant with its environment, contributing to sustainable food and feed production (7). A semisynthetic pathway to produce substrates for the antimalarial artemisinin has been engineered into Escherichia coli (8). The Z-MAPP vaccine, effective against Ebola, comprised recombinant antibodies produced in tobacco plants (9). 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 (10). 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 (10).

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 (11). 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 (12). 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 (13). 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 (14). 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 (14).

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 (12, 15). 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 (16–18). These diameters are consistent with the size and spacing of the six-particle “rosette complexes” that synthesize cellulose microfibrils (19). In cotton fiber cellulose, each glucan chain contains 10,000–14,000 glucosyl units (20, 21), with corresponding lengths of 5–7 μm (22). 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 (23). Electron tomography (24) and atomic force microscopy (25) 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 1.

The 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. 149. 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. 87. 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. 126.

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 (26). 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 (27, 28). 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 (28). 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 (27, 28). Because lignin is structurally heterogeneous, it is difficult to deconstruct to yield intact aromatic molecules as reaction products.

Figure 2.

The 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. 34, as modified in Ref. 27.

Cell-wall compositions vary during plant development, among cell types within a plant, and between species, in response to environmental stimuli (29). 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 (12). 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 (15, 30). The second-generation biorefinery depends on optimizing recovery of glucose and xylose from biomass particles (31). In this context, biomass recalcitrance has been assumed to be the resistance of plant cell walls to enzymatic hydrolysis (15), 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 (32). Many genes encoding the enzymes of lignin biosynthesis have been identified through genetic functional analyses (27, 33–36), enabling engineering of lignin content or composition (27, 28).

In poplar (Populus spp.), disruption of genes encoding certain enzymes of monolignol synthesis, such as C4H (37), 4CL (38, 39), cinnamate 3′-hydroxylase (C3′H) (40, 41), or CCR (42), 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 (40, 43). Down-regulation of CAD to block conversion to monolignol resulted in only modest reduction of lignin in poplar, but enhanced saccharification (44). Two mutations in COMT (45), together with mutations in CAD (46, 47) and 4CL (48), constitute the brown-midrib mutations of sorghum that have enhanced digestibility to improve both ruminant nutrition and sugar yield from saccharification (49). Manipulation of the expression of these genes was sometimes accompanied by reduction of biomass yield (37, 42, 43, 50, 51). Such variable losses might be associated with incomplete xylem stiffening that leads to collapse under water stress (52) or accumulation of inhibitory phenolic substances (53).

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 (53). 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 (54, 55). 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 (56). Introducing alkali-cleavable ferulate esters into the lignin chains resulted in fragmentation into smaller oligomers to reduce recalcitrance (57).

Remarkable flexibility exists in the natural and synthetic monomers that can be polymerized into lignin (28). 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 (58), and 5-hydroxyguaiacyl units are polymerized into 5-hydroxy-G-lignin in other cactus species (59) (Fig. 2). The flavonoid tricin is present in some grass species (60), and hydroxystilbenes, such as resveratrol, also occur as natural substituents of lignin (61). Mutations in CAD, that block the last step in reduction of hydroxycinnamaldehydes to monolignols, incorporate the aldehydes as well as their respective alcohols (44), and mutations in COMT generate 5-hydroxyguaiacyl units that are readily polymerized into lignin at the expense of S-units (55, 62).

Not all phenylpropanoids of the monolignol pathway are incorporated in abundance without growth penalty (63). Down-regulation of CAD introduced more easily cleavable aldehydes (44). When reduced CAD expression was coupled with overexpression of F5H in Arabidopsis, sinapyl aldehyde content was increased, but plants were severely dwarfed (64). A mutation in C3′H (ref8) in Arabidopsis that blocked formation of the G-unit, to produce only the H-unit, also induced severe dwarfism (65), and a similar mutation in poplar caused aberrant accumulation of polyphenols (52). However, secondary mutations in a component of an Arabidopsis transcriptional repressor complex, called Mediator5, rescued both growth and accumulation of H-lignin in the ref8 mutant background (66). 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 (53).

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 (67), ammonia fiber expansion (68), and steam-expansion in water (69), 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 (70). 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 (71). 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 (72). 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 (12). 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 3.

Schematic 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. 126) and features of lignified secondary walls and compound middle lamellae (middle right micrograph, adapted from Ref. 87). Nanoscale structures comprise the cross-bridging of cellulose microfibrils by hemicelluloses in a co-extensive lignin matrix (adapted from Ref. 126) 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 (73–76), 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 (75) or Ni/C (77), 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 (78). 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 (55, 79–81).

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 (82, 83). 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 (84). Pathways that employ lignin as a substrate to replace commodity chemicals have also been envisioned (85, 86). 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 (75). 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 (87). Expression of pectin-degrading enzymes in Arabidopsis increased saccharification yield (88). By contrast, enhanced pectin synthesis from overexpression of homogalacturonan synthase genes decreased saccharification yield in poplar (89), whereas blocking synthesis increased yield (90). 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 (26). 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 (26). 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 (91). However, Arabidopsis plants with high S-lignin content showed disruption of cell-cell adhesion between fiber cells upon treatment with maleic acid (91). 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) (55). Impregnating ferrous iron into biomass, as a co-catalyst with dilute acid, caused the separation of dense aggregates of cellulose microfibrils (92). 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 (93). 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 (94, 95). 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 4.

Rates 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. 55.

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 (55). 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 (96), or ionic liquids, such as 1-butyl-3-methylimidazolium chloride (76, 97), 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 (98). Cellulose swelling was blocked to a large extent by lignin in poplar wood particles, but CDL treatment eliminated this restriction (55). 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 (55). Because of its low boiling point, facile recycling of TFA in a closed system and a continuous-flow cellulose solubilization are possible (98).

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 (78, 83). 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 (82, 83, 99). Controlled fractionation of biomass with consideration of downstream catalytic upgrading provides several value-added streams for xylans to furfural (100), lignin to aromatics and dicarboxylic acids (101), and cellulose to hydroxymethylfurfural (102).

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 (103, 104) and also with carbohydrate-derived monomers (105). 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 (106). 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 (107).

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 (108). More recently, bio-based polyethylene-terephthalate (PET) polymers, made from terephthalates and ethylene glycol, have been widely used to make plastic bottles (109). The entire pathway to polyhydroxybutyrate (PHB) was engineered in cotton almost 25 years ago (110) and more recently in the bioenergy crop switchgrass (111). Routes for the biological synthesis of polyhydroxyurethane have been envisioned (112).

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 (113). The PLAs and their composites were biodegradable, but PETs and PTTs were not (114, 115). 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 (114). By contrast, substantial disintegration of PHA-based plastics, such as PHB, was observed (113). 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 (108). Dissolution of cellulose in solvents such as TFA represents another route to green plastics (116). 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 (117). 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 (78, 118).

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) (119, 120). 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 (121). Microfibrils from ramie plants showed, by small-angle neutron scattering, a periodicity between ordered and disordered domains of only 200–300 residues (122–124). 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 (125). 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 (126), 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 (120, 127).

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 (128), 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 (129, 130), 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) (131). 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 (131). The CNFs contribute strength to the biocomposite, whereas the polymer sheath improves its interfacial stability (131). In addition to fluorescence tagging of monolignols (132, 133), 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 (134). 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 (135). Further surface modifications can be made by attaching propargyl-amino groups through reductive amination to generate reactive groups suitable for these applications (136). Xyloglucan modifications have been extended to functionalize CNCs (137); CNFs have been modified to attach xyloglucan-RGD peptides to enhance cell adhesion and proliferation of human endothelial cells in tissue repair (138).

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 (139), as have those involved in synthesis and regulation of many other hemicellulosic polysaccharides of secondary walls (140, 141). 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 (142). 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 (143), as well as composite films of variable strength with CNFs and galactose-oxidized xyloglucans and guar gums (144). 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 (145), 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 (146, 147), whereas muconic acid can be produced fermentatively and then isomerized and reacted via a Diels–Alder reaction to the diacid (148). 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.