Harnessing xylitol utilization in Escherichia coli for antibiotic-free plasmid and recombinant protein production

Modern genetic medicine such as RNA vaccines are of increasing interest and many of these products require plasmid. There is an increasing interest in developing an antibiotic-free plasmid maintenance system which can potentially speed up clinical trial evaluation and increase product safety. We have previously reported that wildtype E. coli strain cannot utilize xylitol unless a single gene (xylitol dehydrogenase: gutB) was over-expressed. Chemically defined medium formulation was made possible with xylitol as the main carbon source. The present study focused on replacing the antibiotic resistance gene on plasmid with gutB (1056 bp) for maintenance in regular E. coli strains, DH5 alpha and BL21DE3. Four plasmids with different replication origins (pMB1 and pAC) were constructed and selected using the xylitol-based, chemically defined medium. The workflow needs 24 h for colony formation and another 24 h for cell propagation in liquid medium. The plasmid titer and productivity were similar to that obtained using a conventional plasmid and the Luria-Bertani (LB) medium containing an antibiotic. The new plasmid system was also used to produce two recombinant proteins without antibiotics. One of the proteins (a polyethylene terephthalate hydrolase) had similar to 5 times higher specific activity when it is produced using the new system compared with the conventional one. The new plasmid selection/maintenance method reported here could be useful to academic research and industrial bio-manufacturing processes.