Identification of isobutanol response network of E. coli
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ABSTRACT:
ORGANISM(S): Escherichia coli
PROVIDER: GSE13444 | GEO | 2009/11/01
SECONDARY ACCESSION(S): PRJNA110041
REPOSITORIES: GEO
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Project description:Isobutanol has emerged as a potential biofuel due to recent metabolic engineering efforts. Here we used gene expression and transcription factor(TF)-gene interaction data, genetic knockouts, and Network Component Analysis (NCA) to map the isobutanol response network of Escherichia coli under aerobic conditions. A transcriptional response network consisting of 2004 genes/TFs and 2600 interactions was identified. Through further investigation ArcA, Fur, and PhoB were demonstrated to be important mediators of this response. In addition, the ethanol, n-butanol, and isobutanol response networks were compared in order to identify common and distinct toxicity features associated with these three alcohol based biofuels. E. coli was grown aerobically at 37C in minimal MOPS media with 0.2% glucose as the sole carbon source. At mid-log, the cultures were split in half with one half receiving a 1% isobutanol, 1% n-butanol, or 3% ethanol (vol/vol) treatment, while the other half remained unperturbed. After 10 minutes of continued growth cultures were harvested. Total RNA was purified using a Qiagen RNeasy midikit, and labeled indirectly with amino-allyl dUTP. Every experiment had a minimum of 4 biological replicates, each with 2 technical replicates (8 arrays/experiment). Normalized log10 expression ratios were obtained from lcDNA implemented with quality filtering (receptor.seas.ucla.edu/lcDNA; Hyduke DR, Rohlin L, Kao KC, Liao JC. 2003 A software package for cDNA microarray data normalization and assessing confidence intervals. OMICS 7(3):227-234.)
2009-10-31 | E-GEOD-13444 | biostudies-arrayexpress
Project description:Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol, a next-generation biofuel, in Saccharomyces cerevisiae. Here, we explore how two of these strategies, pathway re-localization and redox cofactor-balancing, affect the performance and physiology of isobutanol producing strains. We equipped yeast with isobutanol cassettes which had either a mitochondrial or cytosolic localized isobutanol pathway and used either a redox-imbalanced (NADPH-dependent) or redox-balanced (NADH-dependent) ketoacid reductoisomerase enzyme. We then conducted transcriptomic, proteomic and metabolomic analyses to elucidate molecular differences between the engineered strains. Pathway localization had a large effect on isobutanol production with the strain expressing the mitochondrial localized enzymes producing 3.8-fold more isobutanol than strains expressing the cytosolic enzymes. Cofactor-balancing did not improve isobutanol titers and instead the strain with the redox-imbalanced pathway produced 1.5-fold more isobutanol than the balanced version, albeit at low overall pathway flux. Functional genomic analyses indicated that the poor performances of the cytosolic pathway strains were in part due to a shortage in cytosolic Fe-S clusters, which are required cofactors for the dihydroxyacid dehydratase enzyme. We then demonstrated that this cofactor limitation may be partially recovered by disrupting iron homeostasis with a fra2 mutation, thereby increasing cellular iron levels. The resulting isobutanol titer of the fra2-null strain harboring a cytosolic localized isobutanol pathway outperformed the strain with the mitochondrial localized pathway by 1.3-fold, demonstrating that both localizations can support flux to isobutanol.
2021-10-22 | GSE186126 | GEO
Project description:Isobutanol is considered a potential biofuel and can be produced by genetically modified microorganisms. Saccharomyces cerevisiae inherently produces isobutanol through valine intermediate(s), so it serves as a good host. However, isobutanol's toxicity remains a key obstacle for bioproduction. In our study, we first used image recognition to screen the colony growth of a yeast gene deletion library and inferred that genes involved in tryptophan biosynthesis, ubiquitination, and the pentose phosphate pathway (PPP) contribute to isobutanol tolerance. The importance of tryptophan in yeast's tolerance to isobutanol was confirmed by the recovery of isobutanol tolerance in a tryptophan biosynthesis defective strain by adding exogenous tryptophan. Transcriptomic analysis revealed that amino acid biosynthesis- and transportation-related genes in a tryptophan biosynthesis defective host were up regulated under conditions such as nitrogen starvation. This may explain why ubiquitination for protein degradation was involved for the protein turnover. PPP metabolites and vitamin B1/B6 may serve as precursors and cofactors in tryptophan biosynthesis to enhance isobutanol tolerance. Furthermore, the tolerance mechanism may also be linked to tryptophan metabolism, including the kynurenine pathway and nicotinamide adenine dinucleotide biosynthesis. Both pathways are responsible for cellular redox balance and antioxidative ability and figured out that tryptophan may play a crucial role on isobutanol tolerance. Our study highlights the central role of tryptophan in yeast's isobutanol tolerance and offers new clues for engineering a yeast host with strong isobutanol tolerance.
2021-10-20 | GSE175794 | 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.
2010-08-10 | GSE23526 | 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:This work was primarily focused on removing the main bottleneck for isobutanol production which is the toxicity of isobutanol towards its host and the major reason for its low-level production. Recently, there have been many reports where people have tried various strategies including continuous in-situ product removal from the bioreactor which led to a significant increase in the final product titers. Clearly, it is the intrinsic tolerance levels of the host which decides the end product titers. Importantly, if the host microbe can originally tolerate higher concentrations of toxin (here isobutanol) then it is likely to have a better potential for further improvement of tolerance levels. In our work, we tried to enhance the isobutanol tolerance of wild type NZ9000 using continuous culture. We used the principle of adaptive laboratory evolution to enhance the natural tolerance ability (0.8% isobutanol) of the wild type strain. The strain was cultivated for more than 60 days (>250 generations), while increasing the selection pressure (here isobutanol) gradually in the feed. This finally led to the strain that showed exceptionally higher tolerance (4%) for isobutanol. Transcriptomic analysis also showed fluctuations in gene expression levels from a wide range of categories, precluding any attempt to reverse engineer this phenotype.
2020-07-08 | GSE107996 | GEO
Project description:To elucidate underlying mechanisms responsible for enhanced isobutanol tolerance of the gln3 deletion strain, we performed RNA-seq experiments in biological duplicate to compare the transcriptomic profiles of wild type and gln3 deletion strains grown in SC medium with or without 1.3% (v/v) isobutanol.
2019-11-14 | E-MTAB-8175 | biostudies-arrayexpress
Project description:This project investigated the protein expression pattern in P. polymyxa at different growth stages. The proteomics provided insights into responses of P. polymyxa to isobutanol and revealed a significant metabolic shift caused by alcohol production. These findings were used to evaluate the bottlenecks and further optimize the pathways towards improving product titers.
2024-05-21 | PXD048679 | Pride
Project description:Microarray analysis performed with an evolved strain derived from the industrial strain EthanolRed for accession of the differential expression level of the strain with the biosynthetic pathway for production of isobutanol versus the wild-type strain.
2017-04-10 | E-MTAB-5682 | biostudies-arrayexpress
Project description:Combination of butanol-hyperproducing and hypertolerant phenotypes is essential for developing microbial strains suitable for industrial production of bio-butanol, i.e. one of most promising liquid biofuels. Clostridium cellulovorans is among the microbial strains with the highest potential for direct production of n-butanol from lignocellulosic wastes, a process that would significantly reduce the cost of bio-butanol. However, butanol exhibits higher toxicity compared to ethanol and C. cellulovorans tolerance to this solvent is low. In the present investigation, comparative proteomics was used to study the response of C. cellulovorans to butanol challenge and understand the tolerance mechanisms activated in this condition. The most important response concerned modulation of protein biosynthesis, folding and degradation. Coherent with previous studies on other bacteria, several heat shock proteins (involved in protein quality control) were upregulated. Globally, our data indicates that protein biosynthesis is reduced, likely not to overload heat shock proteins. Several additional metabolic adaptations were triggered by butanol exposure such as the upregulation of V- and F-type ATPases (involved in ATP synthesis/generation of proton motive force), enzymes involved in amino acid biosynthesis, proteins involved in cell envelope re-arrangement and a redistribution of carbon flux through fermentative pathways. These analyses eventually suggested several potential gene targets for metabolic engineering strategies aimed at improving butanol tolerance in C. cellulovorans.
2021-08-13 | PXD024183 | Pride