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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 Zhang1,2*


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-pR 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.



*Corresponding author:           mhzhang@tju.edu.cn
                                               ++86 22 8740 1536
                                               ++86 22 2740 6119

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