Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Dynamic Transcriptional Response of Escherichia coli to Inclusion Body Formation

ABSTRACT: 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 Recombinant cultures producing VP1GFP and GFPCAT in minimal medium (MM) cultures and VP1GFP in ethanol-treated cultures were analyzed. A total of 15 samples were collected. Samples were collected at 5, 20, 40, and 60 minutes post-induction for all induced MM cultures and at 60 minutes post-induction for the induced ethanol-treated cultures. Samples were collected at time 0 and 60 minutes for the uninduced MM and uninduced ethanol-treated cultures. Time 0 of the ethanol-treated VP1GFP cultures consisted of six biological replicates, while the remaining 14 samples were conducted in biological replicates. All biological replicates represent expression data from three independent hybridizations.

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

SUBMITTER: Faraz Baig

PROVIDER: E-GEOD-47732 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Recombinant Escherichia coli cultures are used to manufacture numerous therapeutic proteins and industrial enzymes, where many of these processes use elevated temperatures to induce recombinant protein production. The heat-shock response in wild-type E. coli has been well studied. In this study, the transcriptome profiles of recombinant E. coli subjected to a heat-shock and to a dual heat-shock recombinant protein induction were examined. Most classical heat-shock protein genes were identified as regulated in both conditions. The major transcriptome differences between the recombinant and reported wild-type cultures were heavily populated by hypothetical and putative genes, which indicates recombinant cultures utilize many unique genes to respond to a heat-shock. Comparison of the dual stressed culture data with literature recombinant protein induced culture data revealed numerous differences. The dual stressed response encompassed three major response patterns: induced-like, in-between, and greater than either individual stress response. Also, there were no genes that only responded to the dual stress. The most interesting difference between the dual stressed and induced cultures was the amino acid-tRNA gene levels. The amino acid-tRNA genes were elevated for the dual cultures compared to the induced cultures. Since tRNAs facilitate protein synthesis via translation, this observed increase in amino acid-tRNA transcriptome levels, in concert with elevated heat-shock chaperones, might account for improved productivities often observed for thermo-inducible systems. Most importantly, the response of the recombinant cultures to a heat-shock was more profound than wild-type cultures, and further, the response to recombinant protein induction was not a simple additive response of the individual stresses. The objective of the present work is to gain a better understanding of the heat-shock response in recombinant cultures and how this response might impact recombinant protein production. To accomplish this objective, the transcriptome response of recombinant cultures subjected to a heat-shock and a dual heat-shock recombinant protein induction were analyzed. The transcriptome levels were determined using Affymetrix E. coli Antisense DNA microarrays, such that the entire genome was evaluated. These two transcriptome responses were also compared to recombinant cultures at normal growth temperature that were not over-expressing the recombinant protein and a set of literature recombinant culture data that were chemically induced to over-express the recombinant protein. Additionally, the heat-shock response of the recombinant cultures was compared to the literature report of the heat-shock response in wild-type cultures. The results of the global transcriptome analysis demonstrated that recombinant cultures respond differently to a heat-shock stress than wild-type cultures, where the transcriptome response of the recombinant cultures is further modified by production of a recombinant protein. Experiment Overall Design: The heat-shock and recombinant protein production phases were synchronized to the cell density of 11.5 OD, which is referred to as Sample Time 0. For the heat-shocked cultures, the temperature was increased from 37°C to 50°C over 8 minutes beginning at Sample Time 0. The temperature and duration used in this study are the same conditions used to evaluate the heat-shock in wild-type cultures. The temperature was then decreased from 50°C to 37°C over 4 minutes. For the dual heat-shocked recombinant protein production cultures, 5 mM IPTG was added 8 minutes after Sample Time 0. The unstressed recombinant cultures were conducted similarly, except without the heat-shock or IPTG-addition. Each sample condition was obtained from at least two separate fermentations (two biological replicates). RNA from each biological replicate was purified and processed independently. Prior to hybridization, where only two biological replicates existed, one of the processed samples was divided (two technical replicates), resulting in three separate hybridized chips. The heat-shocked and dual stressed culture samples all consisted of three technical replicates from two biological duplicates. For the unstressed culture samples, triplicate samples were obtained for the 11.5 OD and duplicates for the 14 OD conditions. There were no statistical differences between the 11.5 and 14 OD unstressed samples (p ≤ 0.001). Thus, the unstressed culture transcriptome profile consisted of six technical replicates from five biological replicates and four independent fermentations.

Project description:Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5M-bM-^@M-^Qhydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. Fermentations were carried out in 0.5L bioreactors (Sartorius) containing 0.3L of SynH, SynH lacking osmoprotectants, SynH+LT, or SynH lacking osmoprotectants but containing lignotoxins and cultures were diluted into SynH with initial OD at 0.2, grown anaerobically overnight, and then inoculated into bioreactors to a starting OD600 of 0.2. Two biological replicates (independent cultures) were grown in each medium. RNA samples were obtained at 4 time points, corresponding to exponential (Exp), transitional (Trans), early stationary (Stat1), and late stationary (Stat2) growth phases.

Project description:Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5M-bM-^@M-^Qhydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. E.coli ethanologen strain GLBRCE1 was grown in 3 media, AFEX corn stover hydrolysate (ACSH), synthetic hydrolysate (SynH) and syntetic hydrolysate with added lignotoxins (SynH_LT). Fermentations were carried out in 3 L bioreactors (Applikon Biotechnology) containing 2.45 L of ACSH or SynH media, and cultures were diluted into ACSH or SynH with initial OD at 0.2, grown anaerobically overnight, and then inoculated into bioreactors to a starting OD600 of 0.2. 3 biological replicates (independent cultures) were grown in each medium. RNA samples were obtained at 4 time points, corresponding to exponential (Exp), transitional (Trans), stationary (Stat1) and late stationary (Stat2) growth phases.

Project description:Efficient microbial conversion of lignocellulosic hydrolysates to biofuels is a key barrier to the economically viable deployment of lignocellulosic biofuels. A chief contributor to this barrier is the impact on microbial processes and energy metabolism of lignocellulose-derived inhibitors, including phenolic carboxylates, phenolic amides (for ammonia-pretreated biomass), phenolic aldehydes, and furfurals. To understand the bacterial pathways induced by inhibitors present in ammonia-pretreated biomass hydrolysates, which are less well studied than acid-pretreated biomass hydrolysates, we developed and exploited synthetic mimics of ammonia-pretreated corn stover hydrolysate (ACSH). To determine regulatory responses to the inhibitors normally present in ACSH, we measured transcript and protein levels in an Escherichia coli ethanologen using RNA-seq and quantitative proteomics during fermentation to ethanol of synthetic hydrolysates containing or lacking the inhibitors. Our study identified four major regulators mediating these responses, the MarA/SoxS/Rob network, AaeR, FrmR, and YqhC. Induction of these regulons was correlated with a reduced rate of ethanol production, buildup of pyruvate, depletion of ATP and NAD(P)H, and an inhibition of xylose conversion. The aromatic aldehyde inhibitor 5M-bM-^@M-^Qhydroxymethylfurfural appeared to be reduced to its alcohol form by the ethanologen during fermentation, whereas phenolic acid and amide inhibitors were not metabolized. Together, our findings establish that the major regulatory responses to lignocellulose-derived inhibitors are mediated by transcriptional rather than translational regulators, suggest that energy consumed for inhibitor efflux and detoxification may limit biofuel production, and identify a network of regulators for future synthetic biology efforts. E.coli ethanologen strain GLBRCE1 was grown in 4 media, AFEX corn stover hydrolysate (ACSH), synthetic hydrolysate (SynH), synthetic hydrolysate with added lignotoxins (SynH_LT), or synthetic hydrolysate containing acid or amide lignotoxins only (SynH_Acids_Amides). Fermentations were carried out in 3 L bioreactors (Applikon Biotechnology) containing 2.45 L of ACSH or SynH media, and cultures were diluted into ACSH or SynH with initial OD at 0.2, grown anaerobically overnight, and then inoculated into bioreactors to a starting OD600 of 0.2. Two biological replicates (independent cultures) were grown in each medium. RNA samples were obtained at 6 time points, corresponding to early exponential (Exp1), Mid-exponential (Exp2), Late-exponential (Exp3), transitional (Trans), stationary (Stat1) and late stationary (Stat2) growth phases.

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Dynamic Transcriptional Response of Escherichia coli to Inclusion Body Formation

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