Enhanced electrotransformation of the ethanologen Zymomonas mobilis ZM4 with plasmids

The Zymomonas mobilis ZM4 strain with excellent ethanol‐producing capabilities was the first strain of Z. mobilis, which was sequenced. This strain is resistant to transformation, and no previous study has shown a detailed protocol for electrotransfer of ZM4 with foreign DNA. In this work, many electrical and biological parameters were selected and evaluated in order to optimize the electrotransformation of ZM4. First, improved transformation efficiencies of 11 896, 99, 96 and 5989 transformants/μg DNA were separately achieved with shuttle plasmid pZB21‐mini (3082 bp), pZB21 (5930 bp), pZA22 (6994 bp) and broad‐host‐range vector pBBR1MCS‐2 (5144 bp) all prepared from Escherichia coli JM110. The crucial factors affecting the transformation efficiency included the source of the plasmid (the best strain was ZM4), origin and size of the plasmids, growth phase of the cells (the most ideal phase was early log phase with OD₆₀₀ of 0.3–0.4), the electric field strength (generally 11.75 kV/cm–13.25 kV/cm) and the recovery time (3–24 h). Further, based upon the optimal transformation protocol mentioned above for replicative plasmids in ZM4, (i) the electrotransformation by recombinant plasmid pBBR1MCS‐2‐Pgap‐FLP (6880 bp) was an immediate success with the transformation efficiency 10² transformants/μg DNA; (ii) the site‐specific integration efficiencies (expressed in terms of “per μg of DNA”) of 3–6 integrating transformants was obtained using the integrating plasmid pBR328‐ldhR‐cml‐ldhL (7447 bp). This study will assist genetic and biotechnological research of ZM4 and other Z. mobilis strains by providing information about suitable vectors and a more universal and reliable procedure for introducing DNA into this strain.