Reconstructing a Plant Hemicellulose Biosynthetic Pathway in the Heterologous Fungal Host Pichia Pastoris

The plant cell wall is made of a variety of polysaccharides including the hemicellulose xyloglucan, which is composed of a backbone of β-1,4-linked glucan that is highly substituted with xylose as well as other monosaccharides. Xyloglucan has a wide range of applications including thickening, gelling, and stabilizing agents in the food industry, during weaving of textiles, and as adhesive and binding agents in the pharmaceutical industry.

            In the last decade, the enzymes involved in the biosynthesis of the xyloglucan backbone and substitutions were identified. To provide a testing platform for candidate genes involved in xyloglucan biosynthesis and to investigate the activities of these genes independently from endogenous plant systems, we attempt to reconstruct the plant xyloglucan biosynthetic pathway in the heterologous fungal host Pichia pastoris.

            Our efforts resulted in the production of a glucan backbone in the yeast Pichia pastoris. Subsequent addition of a cytosolic UDP-glucose dehydrogenase as well as a UDP-glucuronic acid decarboxylase made the synthesis of the nucleotide sugar UDP-xylose possible, which is required as a donor substrate for backbone xylosylation. Finally, the addition of a UDP-xylose transporter to transfer UDP-xylose into the lumen of the golgi to the previously mentioned gene resulted in the production of a xylosylated glucan as determined by HPAEC-PAD, Q-TOF and GC-MS analysis.