IS2-mediated overexpression of kfoC in E. coli K4 increases chondroitin-like capsular polysaccharide production.
Transposons are developing molecular tools commonly used for several applications: one of these is the delivery of genes into microorganisms. These mobile genetic elements are characterised by two repeated insertion sequences that flank a sequence encoding one or more orfs for a specific transposase that moves these sequences to other DNA sites. In the present paper, the IS2 transposon of Escherichia coli K4 was modified in vitro by replacing the sequence coding for the transposase with that of the kfoC gene that codes for chondroitin polymerase. KfoC is responsible for the polymerisation of the bacterial capsular polysaccharide whose structure is analogous to that of chondroitin sulphate, a glycosaminoglycan with established and emerging biomedical applications. The recombinant construct was stably integrated into the genome of E. coli K4 by exploiting the transposase from endogenous copies of IS2 in the E. coli chromosome. A significant improvement of the polysaccharide production was observed, resulting in 80 % higher titres in 2.5-L fed-batch cultivations and up to 3.5 g/L in 22-L fed-batch cultures.
Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples, via de Crecchio 7, 80138, Naples, Italy, donatella.cimini@unina2.it.
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D002807:Chondroitin / Q000378:metabolism*; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism*; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic
D004251:DNA Transposable Elements; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins; D002807:Chondroitin; D006602:Hexosyltransferases; C460368:chondroitin polymerase, E coli