Transcription profiling by array of acidogenic chemostat cells of Clostridium acetobutylicum in response to n-butanol
ABSTRACT: Clostridium acetobutylicum is well-known for its butanol production. Butanol toxicity is a major drawback for the generation of high-butanol producing strains. Here, the transcriptional response a steady state, acidogenic (pH 6), phosphate-limited Clostridium acetobutylicum chemostat culture to different levels of n-butanol (0.25-1%) was investigated. For the butanol challenge experiments butanol (1-butanol) was added (a) to the supplying medium and (b) to the culture vessel to guarantee an immediate change in the butanol concentration. Addition of butanol to the culture was timed to match the supply of the new medium through the feedlines. The butanol concentration was increased stepwise in intervals of 66.6 h (5 volume changes) to moderate butanol concentrations of 0.25%, 0.5%, 0.75% and 1% (v/v).
Project description:Clostridium acetobutylicum is a Gram-positive, endospore-forming bacterium that is considered as a strict anaerobe. It ferments sugars to the organic acids acetate and butyrate or shifts to formation of the solvents - ethanol, butanol and acetone. In most bacteria the major regulator of iron homeostasis is Fur (ferric uptake regulator). Analysis of the genome of Clostridium acetobutylicum has revealed three genes encoding Fur-like proteins. The amino acid sequece of one of them showed 70% similarity to the Fur protein of the closely related Bacillus subtilis.<br>Thus, to gain insight into the role of Fur and the mechanisms for maintenance of iron homeostasis in this strict anaerobic organism, we determined its transcriptional profile in response to iron limitation and inactivation of fur.
Project description:Artificial electron carriers have been widely used to shift the solvent ratio towards butanol in acetone-butanol-ethanol (ABE) fermentation of solventogenic clostridia according to decreased hydrogen production. In this study, first insights on the molecular level were gained to explore the effect of methyl viologen addition to cultures of Clostridium acetobutylicum. Employing batch fermentation in mineral salts medium, the butanol:acetone ratio was successively increased from 2.3 to 12.4 on a 100 ml scale in serum bottles and from 1.4 to 16.5 on a 1,300 ml scale in bioreactors, respectively. The latter cultures were used for DNA microarray analyses to provide new information on the transcriptional changes referring to methyl viologen exposure and thus, exhibing gene expression patterns according to the manipulation of the cellular redox balance.
Project description:In this study the transcriptional behavior of the natural solvent producing bacterium Clostridium acetobutylicum was investigated following n butanol stress using DNA microarray analysis. Therefore, a phosphate-limited chemostat culture was established and n-butanol stress (0.9%) was added to acidogenic cells at pH 5.7.
Project description:In this study the transcriptional behavior of the natural solvent producing bacterium Clostridium acetobutylicum was investigated following n-butanol stress using DNA microarray analysis. Therefore, a phosphate-limited chemostat culture was established and n-butanol stress (0.9%) was added to acidogenic cells at pH 5.7.
Project description:The goal of this study is to explore genes that are differentially expressed in E. coli C strains (wt and a butanol-tolerant mutant) after 1-butanol treatment. The butanol-tolerant mutant strain PKH5000 (denoted by 'E' for 'evolved') were derived from KCTC 2571 (wt) (denoted by 'A' for 'ancestral') by proton beam irradiation. 0 and 1 in sample title mean before and after butanol treatment, respectively. All microarray experiments were carried out in triplicate (rep1-3). Probes were spotted in duplicate on separate area of each microarray slide, which produces two GPR files (a and b suffixes).
Project description:Clostridium acetobutylicum is characterized by its acetone-butanol (AB) fermentation which <br>can be reproducibly established under continuous grow conditions in a chemostat. <br>At pH 5.7 cells show typical acidogenic metabolism and mainly produce the acids <br>acetate and butyrate. After lowering and further control the external pH at 4.5 <br>the exponentially growing cells switch towards stable solvent production with the <br>dominating fermentation products acetone and butanol. <br>Here we present a comprehensive comparison of proteome and transcriptome <br>data of continuously growing cells of C. acetobutylicum in a chemostat culture <br>under phosphate limitation at pH 5.7 (acidogenesis) and pH 4.5 (solventogenesis).
Project description:The batch fermentation of Clostridium acetobutylicum is characterized by an acetogenic growth phase during exponential growth when mainly acetate and butyrate are fermentation products. Then, at the end of exponential growth and during stationary phase, the organism switches to solventogenic growth and large amounts of acetone, ethanol and butanol are produced. These growth phases can be studied independent from each other in a phosphate-limited continuous culture. In transcription analysis of continuous cultures using DNA microarrays it became evident that, among others, operons involved in sulfur assimilation are strongly up-regulated during solventogenesis. Using the ClosTron technique we constructed two knock-out mutants in the genes CAC0105 and CAC0930 annotated as involved in sulfate reduction and cysteine biosynthesis. Complementation experiments were carried out with sulfite and cysteine to prove the predicted function. The fermentation experiments of wild type and mutants using phosphate-limited and sulfur-limited continuous culture demonstrated that less sulfur source was consumed in solventogenic phase and the efficiency of cysteine uptake became lower. DNA microarrays were performed to study the difference of transcriptional expression when the wild type was challenged with insufficient sulfur source and the mccB (CAC0930) mutant was inactivated in the continuous culture. The result provided insights into understanding the sulfur metabolism regulatory.
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.)
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. Cultures were grown to OD ~ 0.2 in MRS media (baffled flasks), T = 30 C, 100 rpm. Butanol was then added to the cultures. Samples were harvested 15, 75, and 135 min after butanol addition. Each time point has 3 biological replicates, and dye swaps were incorporated into the microarray experiments.