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Escherichia coli K4 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. The E. coli K4 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·g cdw -1 , according to strain, the type of precursor, the growth phase and the cultivation conditions and their availability dramatically influenced the total CPS produced., (© 2017 The Author(s).)