Oxidative stress sensitivity of engineered Escherichia coli cells with a reduced genome

Abstract

The construction of engineered bacterial cells with a reduced genome allows the investigation of molecular mechanisms that may be cryptic in wild-type strains and derivatives. Previously, a large-scale combined deletion mutant of Escherichia coli that lacked 29.7% of the parental chromosome was constructed by combining large chromosome deletions. In this work, we improved the system for making markerless-chromosomal deletions and obtained mutants with a genome that lacked up to 38.9% of the parental chromosome. Although the large-scale deletion mutants possessed genes needed for resistance to oxidative stress, including superoxide dismutase, catalase, and RpoS, they were sensitive to menadione, which induces reactive oxygen species during stationary phase. Small genome size did not necessarily correlate with greater sensitivity to menadione as several mutants with large deletions were more resistant to menadione. The sensitivity to menadione depended on whether the mutants were grown aerobically or anaerobically, suggesting that the mechanism governing menadione resistance depended on the oxygen tension of the growth medium. Further analysis of the large-scale deletion mutants should help identify the regulatory networks that are important for cellular defense against oxidative stress.

Designing Minimal Genomes Using Whole-Cell Models

“…Minesweeper produced results quickly, within two days the third stage removed 123 genes (a 34% reduction) comparable to current lab-based efforts in other species 3,4,6 . The repeating fourth stage increased the overall number of deletions.…”

“…These are reduced genomes containing only genes essential for life, provided there is a rich growth medium and no external stressors 1,2 . The largest scale efforts in genome minimisation to date include: JCVI-Syn3.0 , a 50% gene reduction of Mycoplasma mycoides 2 ; several strains of Escherichia coli reduced by 38.9% 3 and 35% 4 of their base pairs in-vivo ; an E.coli gene reduction of 77.6% in Saccharomyces cerevisiae 5 ; and two 36% gene reductions of Bacillus subtilis 6 .…”

Systematic identification of synthetic lethal mutations with reduced-genome <i>Escherichia coli</i>: synthetic genetic interactions among <i>yoaA</i>, <i>xthA</i> and <i>holC</i> related to survival from MMS exposure

“…However, reduced-genome strains provide useful tools for systematically identifying cryptic essential genes by taking advantage of their synthetic lethal interactions. To identify such genes, and ultimately a minimal gene set, we previously constructed a series of reduced-genome Escherichia coli strains by combining large-scale chromosomal deletion mutations (Hashimoto et al, 2005;Kato and Hashimoto, 2007;Iwadate et al, 2011). In the present work, using these reduced-genome strains, we identified a synthetic lethality involving the deletion of genes encoding AP endonucleases.…”

Genome Engineering and Modification Toward Synthetic Biology for the Production of Antibiotics

“…loxP sites were inserted into the E. coli genome using λ Red, which relies on the recombination functions of bacteriophage λ. The “minimum genome factory” project launched by Japan has successfully reduced the 4.6‐Mb E. coli K‐12 genome to 2.8‐Mb . The reduced‐genome strain shows enhanced growth and potentially represents a platform for metabolic engineering of higher threonine production.…”

“…The "minimum genome factory" project launched by Japan has successfully reduced the 4.6-Mb E. coli K-12 genome to 2.8-Mb. 51 The reduced-genome strain shows enhanced growth and potentially represents a platform for metabolic engineering of higher threonine production. This strain could also be an important tool for "omics" research.…”