Minimization of the escherichia coli genome using a Tn5-coupled Cre/loxP excision system

Abstract

Many genome sequences of useful industrial microorganisms have been completely sequenced. Based on the results of these genome sequences, functional analyses of microbial genomes have been carried out using comparative genomics and bioinformatics to define functions of new genes and metabolic pathways. These genomic analyses have also been performed in the Escherichia coli which is an important experimental and industrial organism and 620 essential genes and 3,126 nonessential genes have been identified. Of the nonessential genes, E. coli possesses transposable elements, phages, cryptic prophages, motility related genes, horizontally transferred genes, pseudogenes, gene remnants, and damaged operons that would not be needed in the strains designed for biotechnology purposes. These nonessential genes express many unnecessary gene products that represent potential contaminants and metabolic wastes entailed in producing unwanted products that could drive up the cost of product purification and are extra burdens on cell growth. In addition, the genetic instability of the microorganism owing to transposable elements is certainly a problem for producing useful biomaterials and rational strain improvement. Therefore, deletion of these nonessential genes allows us to improve the microorganism for biotechnology purposes. However, conventional methods for deleting many nonessential genes scattered along the E. coli genome have limitations that these methods require either the creation of targeting vectors or complex PCR experiments per each deletion. The need for numerous deletion experiments led us to develop a rapid and efficient deletion procedure. Here, we have developed a powerful genome-engineering tool to delete the nonessential genes of E. coll. We have made two large pools of independent transposon mutants in E. coli using modified Tn5 transposons with a loxP site and two different selection markers and precisely mapped the chromosomal location of 1,000 of these...