Fermentation strategy to enhance plasmid stability during the cultivation of Escherichia coli for the production of recombinant levansucrase

To produce levansucrase, a fructosyltransferase enzyme, in a recombinant Es cherichia coli harboring the levU gene of Zymomonas mobilis, fermentation s trategies were examined in terms of induction methods and plasmid stability . Although the recombinant levansucrase was induced rapidly by IPTG, high i nstability of the plasmid and formation of inclusion bodies were observed. Segregational instability was aggravated by the excretion of beta-lactamase into the culture broth during subculturing, which caused an overgrowth of plasmid-free cells. A combination of methicillin (2, 6-dimethoxyphenyl-peni cillin) and ampicillin to provide selective pressure was effective in preve nting the growth of plasmid-free cells. The population of plasmid-harboring cells was maintained above 95% of the total cells for more than 100 genera tions under this condition. In order to replace IPTG, which is toxic and to o expensive for use in a large scale, D-lactose was tested and found to be favorable as an alternative inducer.