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.
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.
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. C. acetobutylicum cultures were grown, three biological replicates, to mid-exponential phase and then stressed with four levels of butanol (0 mM - No stress; 30 mM - Low stress; 60 mM - Medium stress; & 90 mM - High stress). Samples were collected following the stress at 0, 15, 30, 45, 60 and 75 min, post stress. These sampling times, which are of the order of the doubling time of these cells, were meant to capture largely the direct and immediate impact of these stresses on gene expression. The RNA extracted from two biological replicates were used for microarray hybridization foloowing cDNA generationa nd labelling using Agilent 44K arrays, while the third was used for q-RT-PCR validation. For each stress level, 6 time points with 2 biological replicates and dye swaps (Cy3/Cy5) were prepared for comparison. The hybridization was perfomed against an equal amount of oppositely labeled cDNA from common reference pool prepared using equal amounts of labeled cDNA from all four stress levels.
Project description:An intergenic region found to be enriched from a genomic library under butyrate stress was overexpressed and challenged with butyrate (0.6%). The overexpression strain was compared to the plasmid control to determine the transcriptional changes due to overexpression and butyrate stress.
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 acetate was analyzed. Keywords: stress response
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
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 butyrate was analyzed. Keywords: stress response
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. C. acetobutylicum cultures were grown, three biological replicates, to mid-exponential phase and then stressed with four levels of butyric acid (0 mM - No stress; 30 mM - Low stress; 40 mM - Medium stress; & 50 mM - High stress). Butyric acid was pH adjusted with 10M KOH to match the pH of the cultures, prior to addition. Samples were collected following the stress at 0, 15, 30, 45, 60 and 75 min, post stress. These sampling times, which are of the order of the doubling time of these cells, were meant to capture largely the direct and immediate impact of these stresses on gene expression. The RNA extracted from two biological replicates were used for microarray hybridization foloowing cDNA generation and labelling using Agilent 44K arrays, while the third was used for q-RT-PCR validation. For each stress level, 6 time points with 2 biological replicates and dye swaps (Cy3/Cy5) were prepared for comparison. The hybridization was perfomed against an equal amount of oppositely labeled cDNA from common reference pool prepared using equal amounts of labeled cDNA from all four stress levels.
Project description:Several custom made Agilent arrays were hybridized to select the best 60-mers for the transcriptional profiling of C. acetobutylicum strains. Keywords: Test of 60-mer performance
Project description:Clostridium acetobutylicum naturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineered C. acetobutylicum can be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B-593 (i.e., adh(B-593)) in C. acetobutylicum ATCC 824. To increase the total alcohol titer, a synthetic acetone operon (act operon; adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in the buk gene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged by in situ removal of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.