Isolation of an Escherichia coli K4 kfoC mutant over-producing capsular chondroitin

Abstract

Background: Chondroitin sulphate is a complex polysaccharide having important structural and protective functions in animal tissues. Extracted from animals, this compound is used as a human anti-inflammatory drug. Among bacteria, Escherichia coli K4 produces a capsule containing a non-sulphate chondroitin and its development may provide an efficient and cheap fermentative production of the polysaccharide.

Results: A random N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis was performed on E. coli K4 to isolate mutants showing an increased production of chondroitin. Several mutants were isolated, one of which, here named VZ15, produced about 80% more chondroitin than the wild type E. coli. We found that the mutant has a missense mutation in the codon 313 of kfoC, the gene encoding chondroitin polymerase (K4CP), with a change from arginine to glutamine. A docking analysis to explain the increased productivity of the K4CP enzyme is presented.

Conclusion: The enhanced chondroitin production by the E. coli K4 mutant reported here shows the validity of the strain improvement strategy for more cost-friendly fermentative processes in the production of this pharmaceutically important but so-far expensive polysaccharide.

Figure 1

Colorimetric assay. Colorimetric assay of capsule polysaccharide production performed on wild type E. coli K4 (circled) and some of the selected mutants developing an intense colour (arrows).

Figure 2

Polysaccharide production. Yield for K4-polysaccharide on biomass formed by 15 mutants and the wild type K4 strain. Wild type K4 and VZ15 mutant are evidenced in blue and red, respectively.

Figure 3

Amino acidic sequence. 301-330 amino acidic sequence of K4CP protein from wild type and VZ15 mutant.

Figure 4

Docking analysis. Model complexes UDP-GlcUA/K4CP with Arg-268 in the bent (A) and the extended (B) conformation, UDP-GlcUA/(R313Q)-K4CP with Arg-268 in the bent (C) and the extended (D) conformation, UDP-GalNAc/K4CP (E), and UDP-GalNAc/(R313Q)-K4CP (F). Carbon atoms are colored green, nitrogen atoms blue, oxygen atoms red, phosphorus atoms orange, magnesium ion cyan and hydrogen atoms white. For clarity, only polar hydrogen atoms are shown. H-bonds are shown as yellow dashed lines, whereas other distances are shown as blue dashed lines. Distances are in Å.