title: Enhanced UDP-glucose and UDP-galactose by homologous overexpression of UDP-glucose pyrophosphorylase in Lactobacillus casei.
abstract: UDP-sugars are widely used as substrates in the synthesis of oligosaccharides catalyzed by glycosyltransferases. In the present work a metabolic engineering strategy aimed to direct the carbon flux towards UDP-glucose and UDP-galactose biosynthesis was successfully applied in Lactobacillus casei. The galU gene coding for UDP-glucose pyrophosphorylase (GalU) enzyme in L. casei BL23 was cloned under control of the inducible nisA promoter and it was shown to be functional by homologous overexpression. Notably, about an 80-fold increase in GalU activity resulted in approximately a 9-fold increase of UDP-glucose and a 4-fold increase of UDP-galactose. This suggested that the endogenous UDP-galactose 4-epimerase (GalE) activity, which inter-converts both UDP-sugars, is not sufficient to maintain the UDP-glucose/UDP-galactose ratio. The L. casei galE gene coding for GalE was cloned downstream of galU and the resulting plasmid was transformed in L. casei. The new recombinant strain showed about a 4-fold increase of GalE activity, however this increment did not affect that ratio, suggesting that GalE has higher affinity for UDP-galactose than for UDP-glucose. The L. casei strains constructed here that accumulate high intracellular levels of UDP-sugars would be adequate hosts for the production of oligosaccharides.
authors.affiliation: Laboratorio de Bacterias Lácticas y Probióticos, Instituto de Agroquímica y Tecnología de los Alimentos, CSIC. Av. Agustín Escardino 7, 46980 Paterna, Valencia, Spain.
authors.forename: Jesús
authors.initials: J
authors.lastname: Rodríguez-Díaz
authors.forename: María J
authors.initials: MJ
authors.lastname: Yebra
mesh_terms: D007780:Lacticaseibacillus casei / Q000201:enzymology* / Q000378:metabolism*; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000235:genetics / Q000378:metabolism*; D014531:Uridine Diphosphate Galactose / Q000096:biosynthesis*; D014532:Uridine Diphosphate Glucose / Q000096:biosynthesis*
chemical_list: D014531:Uridine Diphosphate Galactose; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D014532:Uridine Diphosphate Glucose