title: New insight into chondroitin and heparosan-like capsular polysaccharide synthesis by profiling of the nucleotide sugar precursors.
abstract: Escherichia coliK4 and K5 capsular polysaccharides (K4 and K5 CPSs) have been used as starting material for the biotechnological production of chondroitin sulfate (CS) and heparin (HP) respectively. The CPS covers the outer cell wall but in late exponential or stationary growth phase it is released in the surrounding medium. The released CPS concentration was used, so far, as the only marker to connect the strain production ability to the different cultivation conditions employed. Determining also the intracellular UDP-sugar precursor concentration variations, during the bacterial growth, and correlating it with the total CPS production (as sum of the inner and the released ones), could help to better understand the chain biosynthetic mechanism and its bottlenecks. In the present study, for the first time, a new capillary electrophoresis method was set up to simultaneously analyse the UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) and the inner CPS portion, extracted at the same time from the bacterial biomasses; separation was performed at 18°C and 18 kV with a borate-based buffer and detection at 200 nm. TheE. coliK4 and K5 UDP-sugar pools were profiled, for the first time, at different time points of shake flask growths on a glycerol-containing medium and on the same medium supplemented with the monosaccharide precursors of the CPSs: their concentrations varied from 0.25 to 11 μM·gcdw-1, according to strain, the type of precursor, the growth phase and the cultivation conditions and their availability dramatically influenced the total CPS produced.
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania Luigi Vanvitelli, ex Second University of Naples, Via De Crecchio 7, 80138 Naples, Italy odilefrancesca.restaino@unina2.it.
authors.forename: Odile Francesca
authors.initials: OF
authors.lastname: Restaino
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania Luigi Vanvitelli, ex Second University of Naples, Via De Crecchio 7, 80138 Naples, Italy.
authors.forename: Irene
authors.initials: I
authors.lastname: di Lauro
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania Luigi Vanvitelli, ex Second University of Naples, Via De Crecchio 7, 80138 Naples, Italy.
authors.forename: Rosaria
authors.initials: R
authors.lastname: Di Nuzzo
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania Luigi Vanvitelli, ex Second University of Naples, Via De Crecchio 7, 80138 Naples, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: De Rosa
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, University of Campania Luigi Vanvitelli, ex Second University of Naples, Via De Crecchio 7, 80138 Naples, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Schiraldi
mesh_terms: D016667:Bacterial Capsules / Q000378:metabolism*; D053898:Biosynthetic Pathways; D002807:Chondroitin / Q000378:metabolism*; D002809:Chondroitin Sulfates / Q000378:metabolism; D004187:Disaccharides / Q000378:metabolism*; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D007218:Industrial Microbiology; D014530:Uridine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D004187:Disaccharides; C063323:capsular polysaccharide K4; C560363:heparosan; C050673:capsular polysaccharide K5; D014530:Uridine Diphosphate; D006493:Heparin; D002807:Chondroitin; D002809:Chondroitin Sulfates
keywords: Capillary electrophoresis; capsular polysaccharides; chondroitin; heparosan; nucleotide sugars