Dataset Information

Genome-wide dynamic transcriptional profiling in Clostridium beijerinckii NCIMB 8052 using single-nucleotide resolution RNA-Seq.

ABSTRACT: BACKGROUND: Clostridium beijerinckii is a prominent solvent-producing microbe that has great potential for biofuel and chemical industries. Although transcriptional analysis is essential to understand gene functions and regulation and thus elucidate proper strategies for further strain improvement, limited information is available on the genome-wide transcriptional analysis for C. beijerinckii. RESULTS: The genome-wide transcriptional dynamics of C. beijerinckii NCIMB 8052 over a batch fermentation process was investigated using high-throughput RNA-Seq technology. The gene expression profiles indicated that the glycolysis genes were highly expressed throughout the fermentation, with comparatively more active expression during acidogenesis phase. The expression of acid formation genes was down-regulated at the onset of solvent formation, in accordance with the metabolic pathway shift from acidogenesis to solventogenesis. The acetone formation gene (adc), as a part of the sol operon, exhibited highly-coordinated expression with the other sol genes. Out of the > 20 genes encoding alcohol dehydrogenase in C. beijerinckii, Cbei_1722 and Cbei_2181 were highly up-regulated at the onset of solventogenesis, corresponding to their key roles in primary alcohol production. Most sporulation genes in C. beijerinckii 8052 demonstrated similar temporal expression patterns to those observed in B. subtilis and C. acetobutylicum, while sporulation sigma factor genes sigE and sigG exhibited accelerated and stronger expression in C. beijerinckii 8052, which is consistent with the more rapid forespore and endspore development in this strain. Global expression patterns for specific gene functional classes were examined using self-organizing map analysis. The genes associated with specific functional classes demonstrated global expression profiles corresponding to the cell physiological variation and metabolic pathway switch. CONCLUSIONS: The results from this work provided insights for further C. beijerinckii strain improvement employing system biology-based strategies and metabolic engineering approaches.

SUBMITTER: Wang Y

PROVIDER: S-EPMC3395874 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Genome-wide dynamic transcriptional profiling in Clostridium beijerinckii NCIMB 8052 using single-nucleotide resolution RNA-Seq.

Wang Yi Y Li Xiangzhen X Mao Yuejian Y Blaschek Hans P HP

BMC genomics 20120320

<h4>Background</h4>Clostridium beijerinckii is a prominent solvent-producing microbe that has great potential for biofuel and chemical industries. Although transcriptional analysis is essential to understand gene functions and regulation and thus elucidate proper strategies for further strain improvement, limited information is available on the genome-wide transcriptional analysis for C. beijerinckii.<h4>Results</h4>The genome-wide transcriptional dynamics of C. beijerinckii NCIMB 8052 over a ba ...[more]

PMID: 22433311

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Project description:BACKGROUND:Butanol (n-butanol) has high values as a promising fuel source and chemical feedstock. Biobutanol is usually produced by the solventogenic clostridia through a typical biphasic (acidogenesis and solventogenesis phases) acetone-butanol-ethanol (ABE) fermentation process. It is well known that the acids produced in the acidogenic phase are significant and play important roles in the switch to solventogenesis. However, the mechanism that triggers the metabolic switch is still not clear. RESULTS:Sodium butyrate (40 mM) was supplemented into the medium for the ABE fermentation with Clostridium beijerinckii NCIMB 8052. With butyrate addition (reactor R1), solvent production was triggered early in the mid-exponential phase and completed quickly in?<?50 h, while in the control (reactor R2), solventogenesis was initiated during the late exponential phase and took?>?90 h to complete. Butyrate supplementation led to 31% improvement in final butanol titer, 58% improvement in sugar-based yield, and 133% improvement in butanol productivity, respectively. The butanol/acetone ratio was 2.4 versus 1.8 in the control, indicating a metabolic shift towards butanol production due to butyrate addition. Genome-wide transcriptional dynamics was investigated with RNA-Seq analysis. In reactor R1, gene expression related to solventogenesis was induced about 10 hours earlier when compared to that in reactor R2. Although the early sporulation genes were induced after the onset of solventogenesis in reactor R1 (mid-exponential phase), the sporulation events were delayed and uncoupled from the solventogenesis. In contrast, in reactor R2, sporulation genes were induced at the onset of solventogenesis, and highly expressed through the solventogenesis phase. The motility genes were generally down-regulated to lower levels prior to stationary phase in both reactors. However, in reactor R2 this took much longer and gene expression was maintained at comparatively higher levels after entering stationary phase. CONCLUSIONS:Supplemented butyrate provided feedback inhibition to butyrate formation and may be re-assimilated through the reversed butyrate formation pathway, thus resulting in an elevated level of intracellular butyryl phosphate, which may act as a phosphate donor to Spo0A and then trigger solventogenesis and sporulation events. High-resolution genome-wide transcriptional analysis with RNA-Seq revealed detailed insights into the biochemical effects of butyrate on solventogenesis related-events at the gene regulation level.

Project description:BackgroundFurfural is the prevalent microbial inhibitor generated during pretreatment and hydrolysis of lignocellulose biomass to monomeric sugars, but the response of acetone butanol ethanol (ABE) producing Clostridium beijerinckii NCIMB 8052 to this compound at the molecular level is unknown. To discern the effect of furfural on C. beijerinckii and to gain insight into molecular mechanisms of action and detoxification, physiological changes of furfural-stressed cultures during acetone butanol ethanol (ABE) fermentation were studied, and differentially expressed genes were profiled by genome-wide transcriptional analysis.ResultsA total of 5,003 C. beijerinckii NCIMB 8052 genes capturing about 99.7% of the genome were examined. About 111 genes were differentially expressed (up- or down-regulated) by C. beijerinckii when it was challenged with furfural at acidogenic growth phase compared with 721 genes that were differentially expressed (up- or down-regulated) when C. beijerinckii was challenged with furfural at solventogenic growth phase. The differentially expressed genes include genes related to redox and cofactors, membrane transporters, carbohydrate, amino sugar and nucleotide sugar metabolisms, heat shock proteins, DNA repair, and two-component signal transduction system. While C. beijerinckii exposed to furfural stress during the acidogenic growth phase produced 13% more ABE than the unstressed control, ABE production by C. beijerinckii ceased following exposure to furfural stress during the solventogenic growth phase.ConclusionGenome-wide transcriptional response of C. beijerinckii to furfural stress was investigated for the first time using microarray analysis. Stresses emanating from ABE accumulation in the fermentation medium; redox balance perturbations; and repression of genes that code for the phosphotransferase system, cell motility and flagellar proteins (and combinations thereof) may have caused the premature termination of C. beijerinckii 8052 growth and ABE production following furfural challenge at the solventogenic phase.This study provides insights into basis for metabolic engineering of C. beijerinckii NCIMB 8052 for enhanced tolerance of lignocellulose-derived microbial inhibitory compounds, thereby improving bioconversion of lignocellulose biomass hydrolysates to biofuels and chemicals. Indeed, two enzymes encoded by Cbei_3974 and Cbei_3904 belonging to aldo/keto reductase (AKR) and short-chain dehydrogenase/reductase (SDR) families have been identified to be involved in furfural detoxification and tolerance.

Project description:BackgroundSolventogenic clostridia offer a sustainable alternative to petroleum-based production of butanol--an important chemical feedstock and potential fuel additive or replacement. C. beijerinckii is an attractive microorganism for strain design to improve butanol production because it (i) naturally produces the highest recorded butanol concentrations as a byproduct of fermentation; and (ii) can co-ferment pentose and hexose sugars (the primary products from lignocellulosic hydrolysis). Interrogating C. beijerinckii metabolism from a systems viewpoint using constraint-based modeling allows for simulation of the global effect of genetic modifications.ResultsWe present the first genome-scale metabolic model (iCM925) for C. beijerinckii, containing 925 genes, 938 reactions, and 881 metabolites. To build the model we employed a semi-automated procedure that integrated genome annotation information from KEGG, BioCyc, and The SEED, and utilized computational algorithms with manual curation to improve model completeness. Interestingly, we found only a 34% overlap in reactions collected from the three databases--highlighting the importance of evaluating the predictive accuracy of the resulting genome-scale model. To validate iCM925, we conducted fermentation experiments using the NCIMB 8052 strain, and evaluated the ability of the model to simulate measured substrate uptake and product production rates. Experimentally observed fermentation profiles were found to lie within the solution space of the model; however, under an optimal growth objective, additional constraints were needed to reproduce the observed profiles--suggesting the existence of selective pressures other than optimal growth. Notably, a significantly enriched fraction of actively utilized reactions in simulations--constrained to reflect experimental rates--originated from the set of reactions that overlapped between all three databases (P = 3.52 × 10-9, Fisher's exact test). Inhibition of the hydrogenase reaction was found to have a strong effect on butanol formation--as experimentally observed.ConclusionsMicrobial production of butanol by C. beijerinckii offers a promising, sustainable, method for generation of this important chemical and potential biofuel. iCM925 is a predictive model that can accurately reproduce physiological behavior and provide insight into the underlying mechanisms of microbial butanol production. As such, the model will be instrumental in efforts to better understand, and metabolically engineer, this microorganism for improved butanol production.

Project description:BackgroundPlant-based cellulose presents the best source of renewable sugars for biofuel production. However, the lignin associated with plant cellulose presents a hurdle as hydrolysis of this component leads to the production of inhibitory compounds, such as ferulic acid.ResultsThe impacts of ferulic acid, a phenolic compound commonly found in lignin hydrolysates, on the growth, solvent production, and transcriptional responses of Clostridium beijerinckii NCIMB 8052 were determined. Addition of ferulic acid to growing cultures resulted in a decrease in the growth and solvent production by 30% and 25%, respectively, when compared to the control cultures. To better understand the toxicity of this compound, microarray analyses were performed using samples taken from these cultures at three different growth states. Several gene ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were identified showing significant change at each status, including ATP-binding cassette (ABC) transporters, two component system, and oxidoreductase activity. Moreover, genes related with efflux systems and heat shock proteins were also strongly up-regulated. Among these, expression of the groESL operon was induced by more than fourfold and was consequently selected to improve C. beijerinckii tolerance to ferulic acid. Real-time quantitative PCR (RT-qPCR) analysis confirmed that C. beijerinckii harboring the plasmid, pSAAT-ptb_Gro, had a two- to fivefold increased groESL operon expression during growth of these cultures. Moreover, this strain was more tolerant to ferulic acid as the growth of this recombinant strain and its bioconversion of glucose into solvents were both improved.ConclusionsUsing transcriptomics, we identified numerous genes that are differentially expressed when C. beijerinckii cultures were exposed to ferulic acid for varying amounts of time. The operon expressing groESL was consistently up-regulated, suggesting that this gene cluster may contribute to strain tolerance. This was confirmed as recombinant cultures showed both an enhanced growth and solvent yield in the presence of 0.5 g/L ferulic acid.

Dataset Information

Genome-wide dynamic transcriptional profiling in Clostridium beijerinckii NCIMB 8052 using single-nucleotide resolution RNA-Seq.

Publications

Genome-wide dynamic transcriptional profiling in Clostridium beijerinckii NCIMB 8052 using single-nucleotide resolution RNA-Seq.

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