A Markerless Method for Genome Engineering in Zymomonas mobilis ZM4

Abstract

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.

Improving Mobilization of Foreign DNA into Zymomonas mobilis Strain ZM4 by Removal of Multiple Restriction Systems

“…We have also demonstrated the usefulness of our previously published markerless genome modification method ( 5 ) to generate restriction deficient strains by sequentially deleting the four genes encoding the different defense systems from different regions of the genome without introduction of any permanent antibiotic resistance markers. Thus, the absence of any antibiotic resistance cassettes provides an excellent starting point for metabolic strain engineering.…”

“…Primers were obtained from Integrated DNA Technologies (IDT). A Sony MA900 fluorescence activated cell sorter (FACS) was used for sorting nonfluorescent cells from fluorescent cells, and an Azure C600 imager was used for screening of fluorescent colonies as previously described ( 5 ).…”

“…To optimize the metabolism of Z. mobilis and unlock its full potential for industrial production of compounds of interest, introduction of foreign DNA needs to be more reliable and efficient. Recently, we developed a markerless genetic approach to add or remove genes from Z. mobilis strain ZM4 ( 5 ). Here, we used this method to delete genes encoding Z. mobilis restriction systems to test their effects on improving uptake of foreign DNA.…”

Zymomonas diversity and potential for biofuel production

“…Z. mobilis strains have a tendency to flocculate, a trait that can be beneficial for bioprocessing [ 20 , 48 ], but challenging for genetic modification and other laboratory protocols [ 49 ]. Floc formation by Z. mobilis ZM4, CP4 and their flocculating variants is driven by the production of extracellular cellulose fibrils, which become entangled and bind cells into flocs up to 4 mm in length [ 50 , 51 ].…”

“…Cellulose synthesis is required for floc formation in Z. mobilis ZM4, CP4, and flocculating variants and production of cellulose fibrils is performed by the cellulose synthase complex encoded by the bcs operon [ 50 , 51 ]. Strains bearing Tn5 insertions in bcs genes or with the entire operon deleted are not able to form flocs during aerobic growth in rich or minimal media [ 30 , 49 , 53 ]. Several highly flocculent strains have been described previously: PROIMI A1, ATCC 31822 (ZM401) and B-12526, a flocculating derivative of CP4.…”