A Markerless Method for Genome Engineering in ZM4

Metabolic engineering of the biofuel-producing 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 . This is a two-step process that involves homologous recombination of an engineered suicide plasmid bearing 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 () and the operon for cellulose synthase (). In addition, by modifying the plasmid design, we demonstrated targeted insertion of the operon encoding a neurosporene biosynthetic pathway into the genome without addition of any antibiotic resistance genes. We propose this approach will provide an efficient and flexible platform for improved genetic engineering of .