Improved fructosylated chondroitin production by kfoC overexpression in E. coli K4.
Escherichia coli K4 is one of the bacteria expressing a surface polysaccharide, indicated as capsular polysaccharide (K-antigen), showing a chemical structure that resembles that of metabolites commonly used in pharmaceutical applications. In this study we provide evidence that homologous overexpression of the chondroitin polymerase, encoded by the kfoC gene, acts on a potential bottleneck for production of capsular polysaccharide, and increases productivity by 100%. However, we also demonstrate that genetic engineering and scale-up of the production process with E. coli K4 is not straight forward due to genetic instability of recombinant strains, partly overcome by multiple additions of antibiotic throughout fermentation that prove to increase plasmid maintenance inside the cells. A lower resistance to the antibiotic was nevertheless highlighted in the stationary phase suggesting other concomitant causes for plasmid instability. The latter might partly be related to a newly discovered endogenous mobile element that we indicate as pK4EC05. Sequencing and analysis of a 1900 bp fragment of pK4EC05 shows a high percentage of sequence similarity to large conjugative plasmids isolated from Shigella, Salmonella and E. coli strains.
Cimini
Donatella
D
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
De Rosa
Mario
M
Viggiani
Ambra
A
Restaino
Odile F
OF
Carlino
Elisabetta
E
Schiraldi
Chiara
C
D019149:Bioreactors; D015153:Blotting, Western; D002807:Chondroitin / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D006602:Hexosyltransferases / Q000235:genetics* / Q000378:metabolism; D020071:Interspersed Repetitive Sequences / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D017422:Sequence Analysis, DNA
D002807:Chondroitin; D006602:Hexosyltransferases; C460368:chondroitin polymerase, E coli