Evolution of vanillin-tolerant strains of E. coli
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ABSTRACT:
PROVIDER: PRJEB25786 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR2455340_1.fastq.gz | Fastqsanger.gz | |||
| ERR2455340_2.fastq.gz | Fastqsanger.gz | |||
| ERR2455336_1.fastq.gz | Fastqsanger.gz | |||
| ERR2455336_2.fastq.gz | Fastqsanger.gz | |||
| ERR2455337_1.fastq.gz | Fastqsanger.gz |
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Project description:One of the key issues affecting yields of biovanillin in cell factories such as Escherichia coli is product toxicity, the mechanisms of which are poorly understood. To identify targets for engineering improved strains, we have studied mechanisms of vanillin toxicity in E. coli using a two-pronged apparoach: (i) a global proteomic analysis supported by multiple physiological experiments and mutant analyses (ii) adaptive laboratory evolution (ALE) of vanillin tolerance combined with genome sequencing. We identified 147 proteins that exhibited a significant change in abundance in response to vanillin. Upregulated proteins included enzymes capable of converting aldehydes to potentially less toxic compounds; pentose phosphate pathways enzymes, fumarase C and enzymes of the glyoxylate shunt; proteins involved in several key stress responses, in particular oxidative stress; proteins mediating uptake and processing of metal ions. 1H-NMR confirmed that E. coli detoxifies vanillin by reduction to vanillyl alcohol; the aldehyde reductases YqhD and DkgA were highly upregulated by vanillin and the purified enzymes reduced vanillinin an NADPH dependent manner. Vanillin caused accumulation of reactive oxygen species and activated an oxidative stress response through SoxRS, OxyR and MarA pathways. Slow vanillin dependent copper (II) to copper (I) reduction lead to upregulation of the copA gene and growth in the presence of vanillin was hypersensitive to inhibition by copper ions. RT-PCR and mutant growth data suggested AcrD and AaeAB as potential vanillin efflux systems. Vanillin-tolerant strains isolated by ALE had distinct non-synonymous SNPs in the citrate synthase gene gltA, in addition to strain specific mutations in cpdA, rob and marC. One strain had a large ~10 kb deletion including the marRAB region. Purifed variant GltA enzymes all showed higher activity due to a lowered Km for oxaloacetate. Our data provide new understanding and novel gene targets for engineering vanillin-tolerant strains of E. coli, including deletion of the efflux pumps eamA, acrA, and acrB, enhancing oxidative stress defences and NADPH production, improving copper homeostasis and increasing citrate synthase activity using variant enzymes.
2019-05-29 | PXD009242 | Pride
Project description:The vanillin tolerance Saccharomyces cerevisiae was screened and compared intracellular ergosterol levels with several laboratory yeast strains, to study potential relationship between ergosterol contents and vanillin tolerance. S. cerevisiae NBRC1950 was selected as a vanillin tolerant strain. Its ergosterol contents were higher than those of laboratory strains. The results of DNA microarray and quantitative RT-PCR analysis showed that 5 genes involved in ergosterol biosynthesis (ERG28, HMG1, MCR1, ERG5 and ERG7) were up-regulated in NBRC 1950 compared with strain X2180, suggested that high expressions of genes involved in ergosterol biosynthesis may cause for the high ergosterol content in strain NBRC 1950. S. cerevisiae HX strain, which was a high ergosterol content strain derived from X2180, became more tolerant to vanillin compared with the parental strain. It is suggested that high ergosterol contents may be in part responsible for vanillin tolerance. These findings provide a biotechnological basis for the molecular engineering of S. cerevisiae with increased tolerance to vanillin. Experiment Overall Design: Total RNA was extracted from cells in YPD media with shakin by using a hot phenol method. Poly(A)+ RNA was enriched from total RNA by using an Oligotex dT30 (Super) mRNA purification kit (Takara Bio, Ohtsu, Japan). cDNA synthesis, cRNA synthesis, and labeling were performed according to the Affymetrix userâs manual (Affymetrix, Santa Clara, USA). Biotinyated cRNA was fragmented and then used as a probe.Affimetrix Yeast Genome 2.0 arrays (Affymetrix) were used as DNA microarrays. All experiments were done in duplicate independently. Statistical analysis after data acquisition and normalization of expression data were performed using GeneSpring ver.7.3.1 (Agilent Technologies, Palo Alto, CA, USA) based on the gene expression data from two independent experiments. After data transformation to GeneSpring, per-chip normalization to the 50th percentile was performed, and per-gene normalization to the specific samples (X2180 samples) was applied to the per-chip normalized data. Quality control was performed based on experimental confidence levels (each condition in which all samples were present or marginal) and on statistical confidence levels (condition in which P-value of T-test comparisons between X2180 and NBRC 1950 was less than 0.05).
2009-07-04 | E-GEOD-13755 | biostudies-arrayexpress
Project description:Exogenous isopentenol was added to a culture of E. coli and the RNA expression response was measured using Nimblegen arrays. Genes highly upregulated were then subsequently overexpressed to improve the tolerance to isopentenol toxicity. In addition a subset of genes that improved the tolerance were also overexpressed in a producing strain, leading to improved production over the empty vector control. This work is described in Foo et al, Improving microbial bio-gasoline production in E. coli using tolerance engineering (in preparation) Six independent biological replicates were grown in M9 medium. 0.2% v/v isopentenol was added to 3 cultures while 3 cultures received no treatment, and 2.5 hr later total RNA was extracted. One sample from each culture was hybridized.
2013-12-10 | E-GEOD-53138 | biostudies-arrayexpress
Project description:BackgroundYeast strains endowed with robustness towards copper and/or enriched in intracellular Cu might find application in biotechnology processes, among others in the production of functional foods. Moreover, they can contribute to the study of human diseases related to impairments of copper metabolism. In this study, we investigated the molecular and physiological factors that confer copper tolerance to strains of baker's yeasts.ResultsWe characterized the effects elicited in natural strains of Candida humilis and Saccharomyces cerevisiae by the exposure to copper in the culture broth. We observed that, whereas the growth of Saccharomyces cells was inhibited already at low Cu concentration, C. humilis was naturally robust and tolerated up to 1 g · L-1 CuSO4 in the medium. This resistant strain accumulated over 7 mg of Cu per gram of biomass and escaped severe oxidative stress thanks to high constitutive levels of superoxide dismutase and catalase. Both yeasts were then "evolved" to obtain hyper-resistant cells able to proliferate in high copper medium. While in S. cerevisiae the evolution of robustness towards Cu was paralleled by the increase of antioxidative enzymes, these same activities decreased in evolved hyper-resistant Candida cells. We also characterized in some detail changes in the profile of copper binding proteins, that appeared to be modified by evolution but, again, in a different way in the two yeasts.ConclusionsFollowing evolution, both Candida and Saccharomyces cells were able to proliferate up to 2.5 g · L-1 CuSO4 and to accumulate high amounts of intracellular copper. The comparison of yeasts differing in their robustness, allowed highlighting physiological and molecular determinants of natural and acquired copper tolerance. We observed that different mechanisms contribute to confer metal tolerance: the control of copper uptake, changes in the levels of enzymes involved in oxidative stress response and changes in the copper-binding proteome. However, copper elicits different physiological and molecular reactions in yeasts with different backgrounds.
| S-EPMC3276424 | biostudies-literature
Project description:Exogenous isopentenol was added to a culture of E. coli and the RNA expression response was measured using Nimblegen arrays. Genes highly upregulated were then subsequently overexpressed to improve the tolerance to isopentenol toxicity. In addition a subset of genes that improved the tolerance were also overexpressed in a producing strain, leading to improved production over the empty vector control. This work is described in Foo et al, Improving microbial bio-gasoline production in E. coli using tolerance engineering (in preparation)
2013-12-10 | GSE53138 | GEO
Project description:The vanillin tolerance Saccharomyces cerevisiae was screened and compared intracellular ergosterol levels with several laboratory yeast strains, to study potential relationship between ergosterol contents and vanillin tolerance. S. cerevisiae NBRC1950 was selected as a vanillin tolerant strain. Its ergosterol contents were higher than those of laboratory strains. The results of DNA microarray and quantitative RT-PCR analysis showed that 5 genes involved in ergosterol biosynthesis (ERG28, HMG1, MCR1, ERG5 and ERG7) were up-regulated in NBRC 1950 compared with strain X2180, suggested that high expressions of genes involved in ergosterol biosynthesis may cause for the high ergosterol content in strain NBRC 1950. S. cerevisiae HX strain, which was a high ergosterol content strain derived from X2180, became more tolerant to vanillin compared with the parental strain. It is suggested that high ergosterol contents may be in part responsible for vanillin tolerance. These findings provide a biotechnological basis for the molecular engineering of S. cerevisiae with increased tolerance to vanillin.
2009-06-29 | GSE13755 | GEO
Project description:BACKGROUND:Isobutanol is a promising next generation biofuel with demonstrated high yield microbial production, but the toxicity of this molecule reduces fermentation volumetric productivity and final titers. Organic solvent tolerance is a complex, multigenic phenotype that has been recalcitrant to rational engineering approaches. We apply experimental evolution followed by genome resequencing and a gene expression study to elucidate genetic bases on adaptation to exogenous isobutanol stress. RESULTS:The adaptations acquired in our evolved lineages exhibit antagonistic pleiotropy between minimal and rich medium, and appear to be specific to the effects of longer chain alcohols. By examining genotypic adaptation in multiple independent lineages, we find evidence of parallel evolution in hfq, mdh, acrAB, gatYZABCD, and rph genes. Many isobutanol tolerant lineages show reduced rpoS activity, perhaps related to mutations in hfq or acrAB. Consistent with the complex, multigenic nature of solvent tolerance, we observe adaptations in a diversity of cellular processes. Many adaptations appear to involve epistasis between different mutations, implying a rugged fitness landscape for isobutanol tolerance. We observe a trend of evolution targeting post-transcriptional regulation and high centrality nodes of biochemical networks. Collectively, the genotypic adaptations we observe suggest mechanisms of adaptation to isobutanol stress based on remodelling the cell envelope and surprisingly, stress response attenuation. CONCLUSIONS:We have discovered a set of genotypic adaptations that confer increased tolerance to exogenous isobutanol stress. Our results are immediately useful to efforts to engineer more isobutanol tolerant host strains of E. coli for isobutanol production. We suggest that rpoS and post-transcriptional regulators, such as hfq, RNA helicases, and sRNAs may be interesting mutagenesis targets for futurue global phenotype engineering. Two strains (WT strain and G3.2 mutant strain), each with two culture conditions (with and without isobutanol in medium). Three biological replicates for each strain/culture condition. Twelve samples in total.
2010-08-10 | E-GEOD-23526 | biostudies-arrayexpress
Project description:Lignocellulosic biomass is an abundant and renewable resource for biofuels and bio-based chemicals. Vanillin is one of the major phenolic inhibitors in biomass production using lignocellulose. To assess the response of Corynebacterium glutamicum to vanillin stress, a global transcriptional response analysis was performed by using microarray.
2016-08-31 | GSE85949 | GEO
Project description:The selection of bioengineering platform strains and engineering strategies to improve the stress resistance of Saccharomyces cerevisiae remains a pressing need in bio-based chemical production. Thus, a systematic effort to exploit the genotypic and phenotypic diversity to boost yeast’s industrial value is still urgently needed. Here, we analyzed 5400 growth curves obtained from 36 S. cerevisiae strains and comprehensively profiled their resistances against 13 industrially relevant stresses. We observed that bioethanol and brewing strains exhibit higher resistance against acidic conditions, however, plant isolates tend to have wider range of resistance, which may be associated with their metabolome and fluxome signatures in TCA cycle and fatty acid metabolism. By deep genomic sequencing we found that industrial strains have more genomic duplications especially affecting transcription factors, presenting disparate evolutionary paths in comparison to the environmental strains which have more InDels, gene deletions and strain-specific genes. Genome-wide association studies coupled with protein-protein interaction networks uncovered novel genetic determinants of stress resistances. These resistance-related engineering targets and strain rankings provide a valuable source for engineering significantly improved industrial platform strains.</br></br> This metabolomic study of 36 yeast strains measured intra- and extracellular metabolome under standard glucose medium, profiled by GS-MS. This is part of a multi-omic study on yeast strain collection.
2019-01-16 | MTBLS780 | MetaboLights
Project description:Gas-fermenting acetogens, such as Clostridium autoethanogenum, have emerged as promising biocatalysts capable of converting CO and CO2-containing gases into fuels and chemicals relevant for a circular economy. However, functionalities of the majority of genes in acetogens remain uncharacterised, hindering the development of acetogen cell factories through targeted genetic engineering. We previously identified gene targets through adaptive laboratory evolution (ALE) that potentially realise enhanced autotrophic phenotypes in C. autoethanogenum. In this study, we deleted one of the targets – CLAU_0471 (proposed amino acid permease) – with high mutation occurrence in ALE isolates and extensively characterised autotrophic growth of strain RE3 in batch bottle and bioreactor continuous cultures. In addition, we characterized two previously reverse-engineered strains RE1 (deletion of CLAU_3129; putative sporulation transcriptional activator Spo0A) and RE2 (SNP in CLAU_1957; proposed two component transcriptional regulator winged helix family). Strikingly, the strains recovered the superior phenotypes of ALE isolates, including faster autotrophic growth, no need for yeast extract, and robustness in bioreactor operation (e.g. low sensitivity to gas ramping, high biomass, and dilution rates). Notably, RE3 exhibited elevated 2,3-butanediol production while RE1 performed similar to the best-performing previously characterised ALE isolate LAbrini. The three reverse-engineered strains showed similarities in proteome expression and bioinformatic analyses suggest that the targeted genes may be involved in overlapping regulatory networks. Our work provides insights into genotype-phenotype relationships for a better understanding of the metabolism of an industrially-relevant acetogen.
2025-08-12 | PXD062027 | Pride