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

Abstract

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