title: Genetic manipulations restored the growth fitness of reduced-genome Escherichia coli.
abstract: Microbes with smaller genomes would be better chassis for analysis, design, and improvement in the fields of metabolic engineering, synthetic biology, and molecular breeding. To create an Escherichia coli strain with a smaller genome, we used a stepwise genome reduction approach. Beginning with strain MGF-01, which has a genome of 3.62 megabase pairs (Mbp), we generated two E. coli K-12 strains without any insertion sequence (IS), DGF-327 and DGF-298, with reduced genome sizes of 3.27 and 2.98 Mbp, respectively. During the strain construction, intrinsic mutations of ilvG and rph were functionally restored to accelerate initial growth after inoculation. The genomes of the two strains were sequenced, and their structures were confirmed. Both strains showed no auxotrophy, and had better growth fitness, especially in the initial phase, and better cell yield in a rich medium than the wild type K-12 strain. Transcriptome analysis revealed that ibpAB and lon, which encode a heat-shock chaperone and a protease for abnormal proteins, respectively, are down-regulated in DGF strains, compared to the ancestral strains with larger genomes. We concluded that down-regulation of the genes encoding chaperones and proteases is one of the factors that improve the fitness of DGF strains. The DGF strains with fewer genes and better cell yield will be good hosts for applications.
authors.affiliation: Tokyo Research Park, Kyowa Hakko Kirin Co. Ltd., 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan.
authors.forename: Yasutaka
authors.initials: Y
authors.lastname: Hirokawa
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Kawano
authors.forename: Kimie
authors.initials: K
authors.lastname: Tanaka-Masuda
authors.forename: Noriko
authors.initials: N
authors.lastname: Nakamura
authors.forename: Akira
authors.initials: A
authors.lastname: Nakagawa
authors.forename: Mikito
authors.initials: M
authors.lastname: Ito
authors.forename: Hideo
authors.initials: H
authors.lastname: Mori
authors.forename: Taku
authors.initials: T
authors.lastname: Oshima
authors.forename: Naotake
authors.initials: N
authors.lastname: Ogasawara
mesh_terms: D004260:DNA Repair; D015536:Down-Regulation; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D020869:Gene Expression Profiling; D059646:Genome Size; D016680:Genome, Bacterial; D060847:Metabolic Engineering; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D009154:Mutation
chemical_list: D029968:Escherichia coli Proteins; D018832:Molecular Chaperones