Designer biomass for next-generation biorefineries: leveraging recent insights into xylan structure and biosynthesis

Abstract

Xylans are the most abundant noncellulosic polysaccharides in lignified secondary cell walls of woody dicots and in both primary and secondary cell walls of grasses. These polysaccharides, which comprise 20-35% of terrestrial biomass, present major challenges for the efficient microbial bioconversion of lignocellulosic feedstocks to fuels and other value-added products. Xylans play a significant role in the recalcitrance of biomass to degradation, and their bioconversion requires metabolic pathways that are distinct from those used to metabolize cellulose. In this review, we discuss the key differences in the structural features of xylans across diverse plant species, how these features affect their interactions with cellulose and lignin, and recent developments in understanding their biosynthesis. In particular, we focus on how the combined structural and biosynthetic knowledge can be used as a basis for biomass engineering aimed at developing crops that are better suited as feedstocks for the bioconversion industry.

Keywords: Arabinoxylan; Bioindustry; Biosynthesis; Cell wall; Glucuronoxylan; Polysaccharide; Recalcitrance; Xylan.

Fig. 1

Xylan structures from spruce, poplar, and switchgrass secondary walls. Graphical representation of the main structural features of (a) arabinoglucuronoxylan (AGX) from spruce (b) acetylated glucuronoxylan (AcGX) from poplar, and (c) acetylated glucuronoarabinoxylan (AcGAX) from switchgrass. Spruce GX and poplar AcGX contain a distinct glycosidic sequence at their reducing ends, which is absent in switchgrass AcGAX, which often has substituted reducing xylosyl residues at the reducing end [25, 28, 43]. The GlcA and Ara substituents are in even positions and regularly distributed in the main domain of spruce AGX [27, 46]. The substituents in the main domain of Arabidopsis AcGX and poplar are also likely to be evenly distributed [22, 45]. The pattern of distribution of AcGAX substituents in switchgrass secondary walls is still unknown, but they are less branched than the AcGAX in primary walls and other tissue-specific grass xylans (see text for more details)

Fig. 2

Structural features of xylans in bioindustry crops and model organisms. Structural features of xylans from model and industrially relevant plant species. Bars represent detectable amounts of these features described in the literature. Dashed bars represent a lack of analysis describing the presence or absence of these structures. Other structural features not shown may also be present on xylans isolated from these species

Fig. 3

Schematic model of xylan biosynthesis. Xylan biosynthesis takes place in the Golgi lumen. This process requires the generation and transport of several activated nucleotide sugars in addition to both O-acetyl and methyl donors. UDP-Xyl is generated via decarboxylation of UDP-glucuronic acid by UDP-xylose synthase (UXS) in the cytosol, and then transported into the Golgi lumen by UDP-Xyl transporters (UXT) [115]. Synthesis of the xylan backbone is catalyzed by XYS, which is part of a Golgi-localized xylan synthase complex (XSC) that also includes IRX9 and IRX14; however, the roles of the latter enzymes in this process remains enigmatic. UDP-GlcA is transported into the Golgi by a UDP-uronic acid transporter (UUAT) protein [116], and then GUX enzymes catalyze the transfer of GlcA from UDP-GlcA to the xylan backbone, which is subsequently methyl-etherified by GXMT proteins. For the addition of Araf residues, C-4 epimerization of UDP-Xyl to UDP-Arap is carried out by a Golgi-localized UDP-Xyl 4-epimerase (UXE) or cytosolic UDP-glucose 4-epimerases (UGE) [117]. UDP-Arap produced in the Golgi is either used as a substrate in the synthesis of Arap containing polysaccharides such as pectins, or transported back to the cytosol via an unknown process. In the cytosol, UDP-Arap is interconverted to UDP-Araf by UDP-Ara mutases (reversibly glycosylated polypeptide, RGP) [118], and is then transported back into the lumen of the Golgi apparatus by UDP-Araf transporters (UAfT) [119]. XAT enzymes then catalyze the addition of Araf residues to O-3 of the xylan backbone, which is often further substituted by a β-xylosyl residue to O-2 by XAX enzymes. The xylan present in Arabidopsis seed mucilage is also decorated with β-xylosyl residues at O-2, which are added by the xylosyltransferase MUC1. Acetyl donors, such as Acetyl-CoA or an unidentified acetyl donor, are most likely imported into the Golgi lumen by RWA proteins, and then acetylation of the xylan backbone occurs via a number of xylan acetyltransferases (XOAT), which have different catalytic regiospeficities. * Indicates that activity has not been biochemically confirmed

Fig. 4

Models of glucuronoxylan and heparan sulfate biosynthesis. Comparison of proposed models of xylan and heparan sulfate biosynthesis. In bold are enzymes from the families’ common between the two pathways (GT43 and GT47). In heparan sulfate biosynthesis, polysaccharide initiation occurs by the transfer of a xylosyl residue to a protein serine or threonine residue by the enzyme xylosyl transferase 1 (XYLT1) [77]. A linker tetrasaccharide is then synthesized by the enzymes β-1-4 galactosyl transferase 7(β4GalT7), β-1-4 galactosyl transferase 6(β4GalT6) and a GT43 family enzyme Galactosylgalactosylxylosylprotein 3-β-glucuronosyltransferase 3(β3GAT3). Following primer synthesis, the polymer is extended by the GT47/64 heparan synthases, exotosin (EXT) and exotosin-like (EXTL3) proteins, which catalyze the transfer of the repeating segment of glucuronic acid (GlcAp) and N-acetyl glucosamine (GlcNAcp) [77]. This mechanism has similarities to our proposed model for xylan synthesis, where a tetrasaccharide primer may be synthesized while connected to some unknown carrier in the ER/Golgi, potentially in part by GT47 and GT43 family enzymes. This primer is then extended by the GT47 XYS1/IRX10 family of proteins, which most likely function as part of protein complexes that also contain members of GT43 (IRX9, IRX14). The xylan chains are then decorated with sidechains such as acetyl esters and glycosyl units such as (Me) GlcAp