Abstract
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·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.
Highlights
Escherichia coli O5:K4:H4 and O10:K5:H4 capsular polysaccharides (K4 and K5 CPS from Escherichia coli K5 (CPS)) have chondroitin and heparosan-like structures respectively, being made of [→4)-β-D-GlcpA-(1→3)-β-D-GalpNAc-(1→]n, with an extra fructose monosaccharide bounded to the glucuronic acid (GlcA) residue, and of [→4)-β-D-GlcpA-(1→4)-α-D-GlcpNAc-(1→]n, correspondently [1,2]
To address this issue in the present study, a new high-performance capillary electrophoresis (HPCE) method was set up and optimized for the contemporary analysis of UDP-Glc, UDP-Gal, UDP-GalNAc, UDP-GlcNAc, UDP-GlcA and of the inner CPSs as extracted from E. coli K4 and K5 wild-type cells; the nucleotide sugar concentrations were evaluated at different time points of shake flask growths in two different conditions, on a glycerol-based medium and on the same medium supplemented with the CPS monosaccharide precursors [14]
For these reasons we aimed to develop a new, improved, reliable, specific capillary electrophoresis method for the contemporary analysis of UDP-GlcNAc, UDP-GalNAc, UDP-Glc and UDP-GlcA, as extracted from E. coli K4 and K5 cells and that, in addition, could result useful to detect the inner CPS portion in order to correlate the nucleotide sugar pool variations with the total CPS production
Summary
Escherichia coli O5:K4:H4 and O10:K5:H4 capsular polysaccharides (K4 and K5 CPSs) have chondroitin and heparosan-like structures respectively, being made of [→4)-β-D-GlcpA-(1→3)-β-D-GalpNAc-(1→]n, with an extra fructose monosaccharide bounded to the glucuronic acid (GlcA) residue, and of [→4)-β-D-GlcpA-(1→4)-α-D-GlcpNAc-(1→]n, correspondently [1,2]. Developing a good analytical tool to determine the UDP-sugar concentrations and correlate their availability with the kinetic of total CPS production, as sum of the released and inner portion, during the bacterial growth could instead give additional information on the biosynthetic mechanism and how it works in different growth conditions To address this issue in the present study, a new high-performance capillary electrophoresis (HPCE) method was set up and optimized for the contemporary analysis of UDP-Glc, UDP-Gal, UDP-GalNAc, UDP-GlcNAc, UDP-GlcA and of the inner CPSs as extracted from E. coli K4 and K5 wild-type cells; the nucleotide sugar concentrations were evaluated at different time points of shake flask growths in two different conditions, on a glycerol-based medium and on the same medium supplemented with the CPS monosaccharide precursors [14]. The determined inner portion of CPS was summed with the released one in order to obtain the total amount produced at different time points of the bacterial growth and to correlate it with the UDP-sugar precursor availability in the different growth conditions
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