Development of artificial microorganisms by minimizing the escherichia coli genome

Abstract

Numerous genome information was obtained using the result of genome sequences of many microorganisms, functional analyses of the genomes, comparative genomics, and bioinformatics. This information allows us to construct custom-designed microbes useful for human beings. We have selected the transposable elements including IS element, prophage and phage remnant, virulence factors, biofilm, flagella, anaerobic respiration, nonessential protease, K-islands, and horizontally transferred genes as deletion target functions to construct artificial microorganism (custom-designed microbe) with a minimized genome. To delete the selected target regions (genes), we developed efficient genome engineering techniques such as a Tn5-coupled Cre/loxP recombination system, a rapid markerless deletion system, and a bidirectional deletion system. In the Tn5-coupled Cre/loxP recombination system, we quickly and easily deleted many genomic regions without either creation of targeting vectors or complex PCR experiments, using two large loxP-inserted Escherichia coli genome libraries. We also deleted the genomic target regions without remaining of scar using the rapid markerless deletion system, in which the modified linear DNA recombination system, DNA repair mediated by double-strand breakage, and conditional expression of recombination proteins, Red and I-SceI, were combined. In addition, we used the bidirectional deletion system which is a transposon-based deletion technique allows bidirectional random deletion of genomic segments without prior knowledge of which genes are dispensable or prior sequence information. Using these systems, we have individually deleted a total of 1.97 megabase pairs (about 42.5% of the E. coli genome) that does not seem to be essential for cell survival. And we have constructed E. coli with a minimized genome, ME54, in which 54 genomic regions were deleted by cumulative deletions. These deleted regions include large K-specific islands, prophage, ...