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Front Microbiol. 2019 Oct 11;10:2216. doi: 10.3389/fmicb.2019.02216. eCollection 2019.

A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4.

Lal PB1,2, Wells FM1,2, Lyu Y1,3, Ghosh IN1,4, Landick R1,4,5,6, Kiley PJ1,2,5.

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DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI, United States.
Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, United States.
College of Biological and Pharmaceutical Sciences, China Three Gorges University, Yichang, China.
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, United States.
Cell and Molecular Biology Graduate Training Program, University of Wisconsin-Madison, Madison, WI, United States.
Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, United States.


Metabolic engineering of the biofuel-producing Zymomonas mobilis is necessary if we are to unlock the metabolic potential present in this non-model microbe. Manipulation of such organisms can be challenging because of the limited genetic tools for iterative genome modification. Here, we have developed an efficient method for generating markerless genomic deletions or additions in Z. mobilis. This is a two-step process that involves homologous recombination of an engineered suicide plasmid bearing Z. mobilis targeting sequences and a subsequent recombination event that leads to loss of the suicide plasmid and a genome modification. A key feature of this strategy is that GFP expressed from the suicide plasmid allows easy identification of cells that have lost the plasmid by using a fluorescence activated cell sorter. Using this method, we demonstrated deletion of the gene encoding lactate dehydrogenase (ldh) and the operon for cellulose synthase (bcsABC). In addition, by modifying the plasmid design, we demonstrated targeted insertion of the crtIBE operon encoding a neurosporene biosynthetic pathway into the Z. mobilis genome without addition of any antibiotic resistance genes. We propose this approach will provide an efficient and flexible platform for improved genetic engineering of Z. mobilis.


Zymomonas mobilis; biofuels; fluorescence activated cell sorting; genome engineering of a non-model bacterium; green fluorescent protein; recombineering suicide plasmid

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