Project description:We have previously reported that phosphoenolpyruvate carboxykinase(Pck) overexpression under glycolytic conditions enables Escherichia coli to harbor a high intracellular ATP pool resulting in enhanced recombinant protein synthesis and biohydrogen production. To understand possible reasons of the high ATP haboring cell, we carried out transcriptome and metabolic flux analysis.
Project description:The heat-shock response is a cellular protection mechanism against sudden temperature upshifts extensively studied in Escherichia coli. However, the effects of thermal evolution on this response remain largely unknown. In this study, we investigated the early and late physiological and transcriptional responses to temperature upshift in a thermotolerant strain under continuous culture conditions. Adaptive laboratory evolution was performed on a metabolically engineered E. coli strain (JU15), designed for D-lactic acid production, to enable cellular growth and fermentation of glucose at 45 °C in batch cultures. The resulting homofermentative strain, ECL45, successfully adapted to 45 °C in a glucose-mineral medium at pH 7 under non-aerated conditions. The thermal-adapted ECL45 retained the parental strain’s high volumetric productivity and product/substrate yield. Genomic sequencing of ECL45 revealed eight mutations, including one in a non-coding region and six within the coding regions of genes associated with metabolic, transport, and regulatory functions. Transcriptomic analysis comparing the evolved strain with its parental counterpart under early and late temperature upshifts indicated that the adaptation involved an inactive stringent response. This mechanism likely contributes to the strain’s ability to maintain growth capacity at high temperatures.
Project description:We report the effect of oxygenation state in lactose grown escherichia coli producing recombinant proteins. To shed more light on the mechanistic correlation between the uptake of lactose and dissolved oxygen, a comprehensive study has been undertaken with the E. coli BL21 (DE3) strain. Differences in consumption pattern of lactose, metabolites, biomass and product formation due to aerobiosis have been investigated. Transcriptomic profiling of metabolic changes due to aerobic process and microaerobic process during protein formation phase has been studied and the results provide a deeper understanding of protein production in E. coli BL21 (DE3) strains with lactose based promoter expression systems.This study also provides a scientific understanding of escherichia coli metabolism upon oxygen fluctuations.
Project description:To understand the mechanism of isopropanol tolerance of Escherichia coli for improvement of isopropanol production, we performed genome re-sequencing and transcriptome analysis of isopropanol tolerant E. coli strains obtained from parallel adaptive laboratory evolution under IPA stress.
Project description:For over 30 years, serine hydroxamate has been used to chemically stimulate a stringent response in Escherichia coli and other bacteria. These studies have elucidated numerous characteristics of the classical stringent response beyond the simple cellular response to an amino acid shortage, including phospholipid synthesis and protease up-regulation. In this study, the effects of a serine hydroxamate addition on high cell density recombinant E. coli were examined and compared to the effects of recombinant protein production to determine overlaps, as recombinant protein production stress has often been attributed to amino acid shortages. Both the transcriptome and growth characteristics were evaluated and compared. The serine hydroxamate addition profoundly decreased the culture growth rate, whereas, recombinant protein production did not. Conversely, the transcriptome profile of the recombinant E. coli cultures were relatively unaffected by the serine hydroxamate addition, yet recombinant protein production dramatically changed the transcriptome profile. A subset of the classical stringent response genes were effected by the serine hydroxamate addition, whereas, recombinant protein production regulated numerous classical stringent response genes; however, not all. The genes that were regulated by the serine hydroxamate addition include numerous fatty acid synthesis genes, in agreement with altered phospholipids synthesis reports. These results indicate that recombinant protein production and the stringent response have many overlapping responses; however, are far from identical. It was hypothesized that recombinant protein production leads to a stringent response due to the high amino acid synthesis demands related to recombinant protein synthesis. A comparison of the transcriptomes during recombinant protein production and a chemical imposed stringent response would assist with determining what portion of the “metabolic burden” associated with recombinant protein production is due to amino acid shortages. In this study, the transcriptome profiles of recombinant E. coli were examined and compared for the three culture conditions: 1) Normal growth, no external stress; 2) L-serine hydroxamate addition (to mediate a stringent response); and 3) IPTG-induction to produce the recombinant protein chloramphenicol acetyltransferase (CAT). The transcriptome profiles from these three conditions were analyzed using Affymetrix Anti-sense E. coli GeneChip® microarrays.
Project description:DNA microarrays were used to compare the E. coli gene expression response to soluble and insoluble recombinant protein production. The study objective was to characterize the dynamic transcriptional changes that occur as insoluble recombinant protein is produced
Project description:The purpose of this study is to determine whether the presence of pathogenic Escherichia coli in colon is associated with psychiatric disorders.
Project description:Escherichia coli, the common inhabitant of the mammalian intestine, exhibits considerable intraspecies genomic variation, which has been suggested to reflect adaptation to different ecological niches. Also, regulatory trade-offs, e.g., between catabolic versatility and stress protection, are thought to result in significant physiological differences between strains. For these reasons, the relevance of experimental observations made for “domesticated” E. coli strains with regard to the behaviour of this species in its natural environments is often questioned and frequently doubts are raised on the status of E. coli as a defined species. We therefore investigated the variability of important eco-physiological functions such as carbon substrate uptake and breakdown capabilities as well as stress defence mechanisms in the genomes of commensal and pathogenic E. coli strains. Furthermore, eco-physiological properties of environmental strains were compared to standard laboratory strain K-12 MG1655. Catabolic, stress protection, and carbon- and energy source transport operons showed a very low intraspecies variability in 57 commensal and pathogenic E. coli. Environmental isolates adapted to glucose-limited growth in a similar way as E. coli MG1655, namely by increasing their catabolic flexibility and by inducing high affinity substrate uptake systems. Our results indicate that the major eco-physiological properties are highly conserved in the natural population of E. coli. This questions the proposed dominant role of horizontal gene transfer for niche adaptation. Keywords: comparative genomic hybridisation