Genetic manipulations restored the growth fitness of reduced-genome Escherichia coli.

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.

A simple method for multiple modification of the Escherichia coli K-12 chromosome.

We developed a simple method of generating markerless deletions in the Escherichia coli chromosome. The method consists of two recombination events stimulated by lambda Red recombinase. The first recombination replaced a target region with a marker cassette and the second then eliminated the marker cassette. The marker cassette included an antibiotic resistant gene and a negative selection marker (Bacillus subtilis sacB). Since sacB makes E. coli sensitive to sucrose, a markerless deletion strain was successfully selected using its sucrose-resistant phenotype. To stimulate these recombination events, 1-kbp homologous sequences adjacent to the target region were connected to both ends of the marker cassette or connected to each other by PCR. The average efficiency of the recombinations was 24% and 93% respectively. Eliminating the marker cassette with a fragment including an additional sequence, insertion was also possible. This markerless deletion method should be useful in creating a highly modified E. coli chromosome.

Effects of lipoprotein overproduction on the induction of DegP (HtrA) involved in quality control in the Escherichia coli periplasm.

Recent biochemical examination has revealed the presence of at least 90 different lipoproteins in Escherichia coli. Among previously identified lipoproteins, only an outer membrane lipoprotein, NlpE, is known to induce expression of the degP gene upon its overproduction. The degP gene encodes a periplasmic protease, which is thought to be involved in the digestion of unfolded proteins, and is essential for growth at high temperatures. However, it is not completely clear why NlpE overproduction causes degP expression. Moreover, among newly confirmed lipoproteins, there may be others that also induce degP expression. Therefore, we overproduced each of the 90 lipoproteins and examined the level of degP expression as beta-galactosidase activity by using a degP promoter-lacZ fusion. The extent of degP expression caused by NlpE overproduction was dependent on the mode of degP-lacZ fusion. On the other hand, new inner membrane lipoprotein YafY strongly induced degP expression irrespective of the mode of fusion even though the level of overproduced YafY was lower than that of NlpE. The induction of degP expression by YafY overproduction was dependent on the Cpx two-component system. Alteration of the lipoprotein-sorting signals of NlpE and YafY did not abolish the degP induction. However, a YafY derivative possessing the outer membrane signal remained on inner membranes. The non-lipidated derivative of NlpE did not induce degP expression, indicating that membrane anchoring is essential for degP induction. The amino acid sequences of YafY and YfjS, another inner membrane lipoprotein, are highly identical, but overproduction of the latter did not induce degP expression. Construction of various YafY-YfjS chimeric lipoproteins revealed that only a few residues located in the N- and C-terminal regions were important for the induction of DegP.