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Functional Genomic Study of Exogenous n-Butanol Stress in Escherichia coli

ABSTRACT: n-Butanol has been proposed as an alternative biofuel to ethanol, and both Escherichia coli and Saccharomyces cerevisiae have been engineered to produce it. Unfortunately, n-butanol is more toxic than ethanol to these organisms. To understand the basis for its toxicity, cell wide studies were conducted at the transcript, protein and metabolite levels to obtain a global view of the n-butanol stress response. Analysis of the data indicate that n-butanol stress has components common to other stress responses and includes perturbation in respiratory functions (nuo, cyo operons), oxidative stress (sodC, katG, yqhD), heat shock and cell envelope stress (rpoE, clpB, htpG, degP, cpxPR), metabolite transport (malE, opp operon) and biosynthesis. Inducible expression of the yqhD gene was found to improve the host’s tolerance to exogenous n-butanol and confirms the role of this gene in coping with butanol stress. To survey for other potential candidates that may serve to improve host tolerance, mutant strains in several candidates which show changes at the transcript and protein levels were examined for sensitivity during butanol exposure. Chassis engineering based on these cues may be required in a high production titer, butanol-producing host.

ORGANISM(S): Escherichia coli

PROVIDER: GSE16973 | GEO | 2009/12/31

SECONDARY ACCESSION(S): PRJNA117267

REPOSITORIES: GEO

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Functional Genomic Study of Exogenous n-Butanol Stress in Escherichia coli

Project description:n-Butanol has been proposed as an alternative biofuel to ethanol, and both Escherichia coli and Saccharomyces cerevisiae have been engineered to produce it. Unfortunately, n-butanol is more toxic than ethanol to these organisms. To understand the basis for its toxicity, cell wide studies were conducted at the transcript, protein and metabolite levels to obtain a global view of the n-butanol stress response. Analysis of the data indicate that n-butanol stress has components common to other stress responses and includes perturbation in respiratory functions (nuo, cyo operons), oxidative stress (sodC, katG, yqhD), heat shock and cell envelope stress (rpoE, clpB, htpG, degP, cpxPR), metabolite transport (malE, opp operon) and biosynthesis. Inducible expression of the yqhD gene was found to improve the hostâs tolerance to exogenous n-butanol and confirms the role of this gene in coping with butanol stress. To survey for other potential candidates that may serve to improve host tolerance, mutant strains in several candidates which show changes at the transcript and protein levels were examined for sensitivity during butanol exposure. Chassis engineering based on these cues may be required in a high production titer, butanol-producing host. This comparison is between E. coli DH1 cells treated with 0.8% n-butanol and untreated cells at 0, 30, 80, and 195 minutes after addition. 3 biological replicates were grown, total RNA was extracted, labeled, and hybridized on 3 slides for each time point. http://www.microbesonline.org/cgi-bin/microarray/viewExp.cgi?expId=1266

2010-05-16 | E-GEOD-16973 | biostudies-arrayexpress

Metabolite stress response to butanol in Clostridium acetobutylicum ATCC 824

Project description:Clostridium acetobutylicum is a Gram positive, endospore forming firmicute that has been known as the model organims for ABE (acetone-butanol-ethanol) fermentation. With its ability to consume a wide variety of substrates, C. acetobutylicum carries out a biphasic ABE fermentation, which consists of the acidogenic growth phase with the formation of butyric acid and acetic acid, followed by the solventogenic stationary phase with the formation of acetone, butanol and ethanol, characterised by the reassimilation of acids. The production butanol is of renewed ineterest both as a potential biofuel and bulk chemical production. Both butanol and butyrate posses toxic characteristic and here, we focus on understanding and modeling the stress response of C. acetobutylicum to one of the two important toxic metabolites: butanol.

2013-08-20 | GSE48031 | GEO

Functional identification of butanol-tolerant genes mediated by the transcriptional factor Rob in Escherichia coli

Project description:Butanol is a bulk chemical feedstock and a promising fuels. Microbial production of butanol is challenging primarily because of its toxicity and low titer of production. Transcript regulator factor Rob plays an important role in butanol-tolerant functional revealed by our previous study. In this study, the mutant strain DTrob (AT686-687 deletion in rob gene) could tolerate 1.25 %(v/v) butanol. And the per unit intracellular butanol concentration and transcriptome of wild-type and DTrob were further compared to understand the regulation mechanism of Rob for butanol tolerance. A total of 285 DEGs (differentially expression genes) were identified by bioinformatic analysis, and they may function in transport and localization according to the GO functional annotations and the functional annotation of gene product in NCBI database. Eighteen DEGs representing different functional categories were selected for real-time quantitative PCR (qPCR) for confirming the dependability of RNA-seq data. The deletion strains of glgS or yibT gene, which were down-regulated by Rob, had butanol-toleranct characteristic, indicating that the deletion or downregulated expression of the two genes down-regulated by Rob could improve the butanol tolerance. This study reveals the transcript regulator factor Rob could regulate and control the expression of the butanol tolerant genes, and its inactivation would result in an improved butanol tolerance.

2019-09-17 | GSE120032 | GEO

Stress-driven feedback regulation of multiple tolerant genes improves xylose fermentation of Saccharomyces cerevisiae

Project description:The modification of the The modification of the tolerance of xylose-fermenting yeast is an urgent issue for improving ethanol production. In this study, multiple genes involving in superoxide dismutase, glutathione biosynthesis, NADPH regeneration and acetic acid degradation were overexpressed using stress-induced promoters, which is selected from the transcriptome data. Stress-induced promoters were used to realize the feedback control of the tolerant genes, which can ultimately improve the tolerance and ethanol production. We reported the stress-induced promoters for overexpressing tolerant genes and increasing yeast tolerance in a feedback manner

2022-01-01 | GSE140075 | GEO

Metabolite stress response to butyric acid in Clostridium acetobutylicum ATCC 824

Project description:Clostridium acetobutylicum is a Gram positive, endospore forming firmicute that has been known as the model organims for ABE (acetone-butanol-ethanol) fermentation. With its ability to consume a wide variety of substrates, C. acetobutylicum carries out a biphasic ABE fermentation, which consists of the acidogenic growth phase with the formation of butyric acid and acetic acid, followed by the solventogenic stationary phase with the formation of acetone, butanol and ethanol, characterised by the reassimilation of acids. The production butanol is of renewed ineterest both as a potential biofuel and bulk chemical production. Both butanol and butyric acid posses toxic characteristic and here, we focus on understanding and modeling the stress response of C. acetobutylicum to one of the two important toxic metabolites: butyric acid.

2013-08-20 | GSE48039 | GEO

Clostridium cellulovorans proteomic responses to butanol stress

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

Clostridium acetobutylicum ATCC 824 butanol stress (50 mM)

Project description:Metabolite accumulation has pleiotropic, including toxic, effects on cellular physiology, but such effects are not well understood at the genomic level. Using DNA microarrays, the Clostridium acetobutylicum transcriptional stress response to butanol was analyzed. Keywords: stress response

2007-12-29 | GSE5008 | GEO

Ethanol cellular defense induce unfolded protein response in yeast

Project description:Ethanol is a valuable industrial product and a common metabolite used by many cell types. However, this molecule produces high levels of cytotoxicity affecting cellular performance at several levels. In the presence of ethanol, cells must adjust some of their components, such as the membrane lipids to maintain homeostasis. In the case of microorganism as Saccharomyces cerevisiae, ethanol is one of the principal products of their metabolism and is the main stress factor during fermentation. Although many efforts have been made, mechanisms of ethanol tolerance are not fully understood and very little evidence is available to date for specific signaling by ethanol in the cell. This work studied two Saccharomyces cerevisiae strains, CECT10094 and Temohaya-26, isolated from flor wine and traditional fermentations respectively, which differ in ethanol tolerance, in order to understand the molecular mechanisms underlying the ethanol stress response and the reasons for different ethanol tolerance. The transcriptome was analyzed after ethanol stress and, among others, an increased activation of genes related with the unfolded protein response (UPR) and its transcription factor, Hac1p, was observed in the tolerant strain CECT10094. We observed that this strain also resist more UPR agents than Temohaya-26 and the UPR-ethanol stress correlation was corroborated observing growth of 15 more strains and discarding UPR correlation with other stresses as thermal or oxidative stress. Furthermore, higher activation of UPR pathway in the tolerant strain CECT10094 was observed using a UPR mCherry reporter. Finally, we observed UPR activation in response to ethanol stress in other S. cerevisiae ethanol tolerant strains as the wine strains T73 and EC1118.

2016-02-03 | GSE44863 | GEO

A transcriptional regulator Sll0794 regulates tolerance to biofuel ethanol in photosynthetic Synechocystis sp. PCC 6803

Project description:To improve ethanol production directly from CO2 in photosynthetic cyanobacterial systems, one key issue that needs to be addressed is the low ethanol tolerance of cyanobacterial cells. Our previous proteomic and transcriptomic analyses found that several regulatory proteins were up regulated by exogenous ethanol in Synechocystis sp. PCC 6803. In this study, through tolerance analysis of the gene disruption mutants of the up-regulated regulatory genes, we uncovered that one transcriptional regulator, Sll0794, was related directly to ethanol tolerance in Synechocystis. Using a quantitative iTRAQ-LC-MS/MS proteomics approach coupled with quantitative real-time reverse transcription-PCR (RT-qPCR), we further determined the possible regulatory network of Sll0794. The proteomic analysis showed that in the ∆sll0794 mutant grown under ethanol stress a total of 54 and 87 unique proteins were down- and up-regulated, respectively. In addition, electrophoretic mobility shift assays (EMSAs) demonstrated that the Sll0794 transcriptional regulator was able to bind directly to the upstream regions of sll1514, slr1512 and slr1838, which encode a 16.6 kDa small heat shock protein, a putative sodium-dependent bicarbonate transporter and a carbon dioxide concentrating mechanism protein CcmK, respectively. The study provided a proteomic description of the putative ethanol-tolerance network regulated by the sll0794 gene, and revealed new insights on the ethanol-tolerance regulatory mechanism in Synechocystis. As the first regulatory protein discovered related to ethanol tolerance, the gene may serve as a valuable target for transcription machinery engineering to further improve ethanol tolerance in Synechocystis.

2014-09-26 | PXD001266 | Pride

Butanol Challenge of L. brevis

Project description:The presence of anti-microbial phenolic compounds, such as the model compound ferulic acid, in biomass hydrolysates poses significant challenges to the widespread use of biomass in conjunction with whole cell biocatalysis or fermentation. Biofuel toxicity must also be overcome to allow for efficient production of next generation biofuels such as butanol, isopropanol, and others for widespread usage. Currently, these inhibitory compounds must be removed through additional downstream processing or sufficiently diluted to create environments suitable for most industrially important microbial strains. This study explores the high ferulic acid and n-butanol tolerance in Lactobacillus brevis (L. brevis), a lactic acid bacteria often found in fermentation processes, by global transcriptional response analysis. The transcriptional profile of L. brevis under ferulic acid and butanol stress reveals that the presence of ferulic acid primarily triggers the expression of membrane proteins to counteract ferulic acid induced changes in membrane fluidity and ion leakage. In contrast to the ferulic acid stress response, butanol addition to growing cultures uniquely induced the entire fatty acid synthesis pathway in the midst of a generalized stress response. Overexpression of the rate-limiting acetyl-CoA carboxylase subunits (AccABCD) in E. coli to increase lipid synthesis had no effect on butanol tolerance, suggesting that additional engineering is necessary to produce sufficient levels of appropriate fatty acids to confer butanol tolerance. Several promising routes for understanding both phenolic acid and butanol tolerance have been identified based upon these findings. These insights may be used to guide further engineering of model industrial organisms to better tolerate both classes of inhibitors in processed biomass used for biofuel production.

2011-12-31 | GSE24944 | GEO

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