Overexpression of UDP-glucose dehydrogenase in Escherichia coli results in decreased biosynthesis of K5 polysaccharide.
The Escherichia coli K5 capsular polysaccharide (glycosaminoglycan) chains are composed of the repeated disaccharide structure: -GlcAbeta1,4-GlcNAcalpha1,4-(where GlcA is glucuronic acid and GlcNAc is N-acetyl-D-glucosamine). The GlcA, present in most glycosaminoglycans, is donated from UDP-GlcA, which, in turn, is generated from UDP-glucose by the enzyme UDP-glucose dehydrogenase (UDPGDH). The formation of UDP-GlcA is critical for the biosynthesis of glycosaminoglycans. To investigate the role of UDPGDH in glycosaminoglycan biosynthesis, we used K5 polysaccharide biosynthesis as a model. E. coli was transformed with the complete gene cluster for K5 polysaccharide production. Additional transformation with an extra copy of UDPGDH resulted in an approx. 15-fold increase in the in vitro UDPGDH enzyme activity compared with the strain lacking extra UDPGDH. UDP-GlcA levels were increased 3-fold in overexpressing strains. However, metabolic labelling with [14C]glucose showed, unexpectedly, that overexpression of UDPGDH lead to decreased formation of K5 polysaccharide. No significant difference in the K5 polysaccharide chain length was observed between control and overexpressing strains, indicating that the decrease in K5-polysaccharide production most probably was due to synthesis of fewer chains. Our results suggest that K5-polysaccharide biosynthesis is strictly regulated such that increasing the amount of available UDP-GlcA results in diminished K5-polysaccharide production.
Roman
Elisabet
E
Department of Medical Biochemistry and Microbiology, University of Uppsala, BMC Box 582, SE-751 23 Uppsala, Sweden.
Roberts
Ian
I
Lidholt
Kerstin
K
Kusche-Gullberg
Marion
M
D016667:Bacterial Capsules / Q000096:biosynthesis / Q000235:genetics; D003864:Depression, Chemical; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis* / Q000737:chemistry; D014162:Transfection; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics; D014535:Uridine Diphosphate Glucuronic Acid / Q000737:chemistry
D029968:Escherichia coli Proteins; D011135:Polysaccharides, Bacterial; D014535:Uridine Diphosphate Glucuronic Acid; C050673:capsular polysaccharide K5; C097522:KfiD protein, E coli; D014533:Uridine Diphosphate Glucose Dehydrogenase