Reconstruction of de novo pathway for synthesis of UDP-glucuronic acid and UDP-xylose from intrinsic UDP-glucose in Saccharomyces cerevisiae.
UDP-D-glucuronic acid and UDP-D-xylose are required for the biosynthesis of glycosaminoglycan in mammals and of cell wall polysaccharides in plants. Given the importance of these glycans to some organisms, the development of a system for production of UDP-D-glucuronic acid and UDP-D-xylose from a common precursor could prove useful for a number of applications. The budding yeast Saccharomyces cerevisiae lacks an endogenous ability to synthesize or consume UDP-D-glucuronic acid and UDP-D-xylose. However, yeast have a large cytoplasmic pool of UDP-D-glucose that could be used to synthesize cell wall beta-glucan, as a precursor of UDP-D-glucuronic acid and UDP-D-xylose. Thus, if a mechanism for converting the precursors into the end-products can be identified, yeast may be harnessed as a system for production of glycans. Here we report a novel S. cerevisiae strain that coexpresses the Arabidopsis thaliana genes UGD1 and UXS3, which encode a UDP-glucose dehydrogenase (AtUGD1) and a UDP-glucuronic acid decarboxylase (AtUXS3), respectively, which are required for the conversion of UDP-D-glucose to UDP-D-xylose in plants. The recombinant yeast strain was capable of converting UDP-D-glucose to UDP-D-glucuronic acid, and UDP-D-glucuronic acid to UDP-D-xylose, in the cytoplasm, demonstrating the usefulness of this yeast system for the synthesis of glycans. Furthermore, we observed that overexpression of AtUGD1 caused a reduction in the UDP-D-glucose pool, whereas coexpression of AtUXS3 and AtUGD1 did not result in reduction of the UDP-D-glucose pool. Enzymatic analysis of the purified hexamer His-AtUGD1 revealed that AtUGD1 activity is strongly inhibited by UDP-D-xylose, suggesting that AtUGD1 maintains intracellular levels of UDP-D-glucose in cooperation with AtUXS3 via the inhibition of AtUGD1 by UDP-D-xylose.
Oka
Takuji
T
Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
Jigami
Yoshifumi
Y
D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D007700:Kinetics; D013058:Mass Spectrometry; D008954:Models, Biological; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism*; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D014535:Uridine Diphosphate Glucuronic Acid / Q000096:biosynthesis*; D014540:Uridine Diphosphate Xylose / Q000096:biosynthesis*
D029681:Arabidopsis Proteins; D011994:Recombinant Proteins; D014535:Uridine Diphosphate Glucuronic Acid; D014540:Uridine Diphosphate Xylose; C513668:UGD1 protein, Arabidopsis; D014533:Uridine Diphosphate Glucose Dehydrogenase; D002262:Carboxy-Lyases; C023257:UDPglucuronate decarboxylase; D014532:Uridine Diphosphate Glucose