An update on xylan structure, biosynthesis, and potential commercial applications.

•Xylan is an abundant carbohydrate component of plant cell walls that is vital for proper cell wall structure and vascular tissue development.•Xylan structure is known to vary between different tissues and species.•The role of xylan in the plant cell wall is to interact with cellulose, lignin, and hemicelluloses.•Xylan synthesis is directed by several types of Golgi-localized enzymes.•Xylan is being explored as an eco-friendly resource for diverse commercial applications.

Enzymatic Synthesis of Xylan Microparticles with Tunable Morphologies.

Xylans are a diverse family of hemicellulosic polysaccharides found in abundance within the cell walls of nearly all flowering plants. Unfortunately, naturally occurring xylans are highly heterogeneous, limiting studies of their synthesis and structure-function relationships. Here, we demonstrate that xylan synthase 1 from the charophyte alga Klebsormidium flaccidum is a powerful biocatalytic tool for the bottom-up synthesis of pure ß-1,4 xylan polymers that self-assemble into microparticles in vitro. Using uridine diphosphate-xylose (UDP-xylose) and defined saccharide primers as substrates, we demonstrate that the shape, composition, and properties of the self-assembling xylan microparticles could be readily controlled via the fine structure of the xylan oligosaccharide primer used to initiate polymer elongation. Furthermore, we highlight two approaches for bottom-up and surface functionalization of xylan microparticles with chemical probes and explore the susceptibility of xylan microparticles to enzymatic hydrolysis. Together, these results provide a useful platform for structural and functional studies of xylans to investigate cell wall biosynthesis and polymer-polymer interactions and suggest possible routes to new biobased materials with favorable properties for biomedical and renewable applications.

Rational enzyme design for controlled functionalization of acetylated xylan for cell-free polymer biosynthesis.

Xylan O-acetyltransferase 1 (XOAT1) is involved in O-acetylating the backbone of hemicellulose xylan. Recent structural analysis of XOAT1 showed two unequal lobes forming a cleft that is predicted to accommodate and position xylan acceptors into proximity with the catalytic triad. Here, we used docking and molecular dynamics simulations to investigate the optimal orientation of xylan in the binding cleft of XOAT1 and identify putative key residues (Gln445 and Arg444 on Minor lobe & Asn312, Met311 and Asp403 on Major lobe) involved in substrate interactions. Site-directed mutagenesis coupled with biochemical analyses revealed the major lobe of XOAT1 is important for xylan binding. Mutation of single key residues yielded XOAT1 variants with various enzymatic efficiencies that are applicable to one-pot synthesis of xylan polymers with different degrees of O-acetylation. Taken together, our results demonstrate the effectiveness of computational modeling in guiding enzyme engineering aimed at modulating xylan and redesigning plant cell walls.