Optimization of the enzymatic pathway for UDP-xylose biosynthesis
The plant cell wall is of interest for agriculture and biofuel production; understanding the composition and biosynthesis of plant cell walls is critical for improving the viability of biorenewable energy as a replacement for traditional energy sources. Hemicellulose is a common cell wall constituent that hasn’t undergone much study and contains a significant amount of xylosylated compounds including xylan and xyloglucan. Elucidating properties of xylosylated hemicellulose via characterization of hemicellulosic xylosyltransferases requires the substrate UDP-xylose (UDPX) for in vitro enzymatic assays. The high cost and a single producer of UDPX limits its practical use, while labeled UDPX isn’t commercially available. Therefore, this project sought to synthesize standard and labeled UDPX from the readily available precursor, UDP-glucose (UDPG), in an efficient, cost-effective manner. The efficient one-pot synthesis of UDPX using recombinant UDPglucose dehydrogenase (UGD) and UDP-xylose synthase (UXS) is the working goal. Currently, the kinetics of UGD are characterized and the UGD assay conditions have been optimized to 90% substrate conversion efficiency. Activity of the UXS assay was confirmed and the methodology for analyzing UDPX production is set. The project continues to optimize the UXS assay conditions and will follow up by combining the two assays.