Construction of an Unmarked Zymomonas mobilis Mutant Using a Site-Specific FLP Recombinase

Shao-Lan Zou¹, Jie-Fang Hong¹, Cui Wang¹, Xin Jing¹ and Min-Hua Zhang^1,2*

¹Biomass Conversion Laboratory, Tianjin R&D Center For Petrochemical Technology, Tianjin University, Tianjin, 300072, P.O. Box 6888, PR China
²State Key Laboratory of Engines, Tianjin University, Tianjin, 300072, P.O. Box 6888, PR China

Article history:
Received March 6, 2011
Accepted December 12, 2011

Key words:
Zymomonas mobilis, homologous recombination, flippase expression, transformation, unmarked mutants

Summary:
Flippase expression was carried out in Zymomonas mobilis strain ZM4. The FRT-flanked selection marker gene was first integrated into the ZM4 chromosome by homologous recombination. The Saccharomyces cerevisiae flp gene was then introduced under the control of the ZM4 gap gene promoter (Pgap, encoding glyceraldehyde-3-phosphate dehydrogenase) or the λ bacteriophage cI₈₅₇-p_R contained in the broad-host-range cloning vector pBBR1- MCS-2. This study demonstrated that flp was expressed and that the deletion frequency of the FRT-flanked marker gene was very high (approx. 100 %). In addition, the flp gene expression vector could be conveniently removed from the resulting unmarked Z. mobilis mutants by serially transferring the cells three times into antibiotic-free medium, thereby establishing an efficient method for constructing unmarked Z. mobilis mutants.

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