Evidence of mixotrophic carbon-capture by n-butanol-producer Clostridium beijerinckii.
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SUBMITTER: Sandoval-Espinola WJ
PROVIDER: S-EPMC5630571 | biostudies-literature | 2017 Oct
REPOSITORIES: biostudies-literature
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Evidence of mixotrophic carbon-capture by n-butanol-producer Clostridium beijerinckii.
Sandoval-Espinola W J WJ Chinn M S MS Thon M R MR Bruno-Bárcena J M JM
Scientific reports 20171006 1
Recent efforts to combat increasing greenhouse gas emissions include their capture into advanced biofuels, such as butanol. Traditionally, biobutanol research has been centered solely on its generation from sugars. Our results show partial re-assimilation of CO<sub>2</sub> and H<sub>2</sub> by n-butanol-producer C. beijerinckii. This was detected as synchronous CO<sub>2</sub>/H<sub>2</sub> oscillations by direct (real-time) monitoring of their fermentation gasses. Additional functional analysis ...[more]
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Project description:BACKGROUND:Thinning supplies of natural resources increase attention to sustainable microbial production of bio-based fuels. The strain Clostridium beijerinckii NRRL B-598 is a relatively well-described butanol producer regarding its genotype and phenotype under various conditions. However, a link between these two levels, lying in the description of the gene regulation mechanisms, is missing for this strain, due to the lack of transcriptomic data. RESULTS:In this paper, we present a transcription profile of the strain over the whole fermentation using an RNA-Seq dataset covering six time-points with the current highest dynamic range among solventogenic clostridia. We investigated the accuracy of the genome sequence and particular genome elements, including pseudogenes and prophages. While some pseudogenes were highly expressed, all three identified prophages remained silent. Furthermore, we identified major changes in the transcriptional activity of genes using differential expression analysis between adjacent time-points. We identified functional groups of these significantly regulated genes and together with fermentation and cultivation kinetics captured using liquid chromatography and flow cytometry, we identified basic changes in the metabolism of the strain during fermentation. Interestingly, C. beijerinckii NRRL B-598 demonstrated different behavior in comparison with the closely related strain C. beijerinckii NCIMB 8052 in the latter phases of cultivation. CONCLUSIONS:We provided a complex analysis of the C. beijerinckii NRRL B-598 fermentation profile using several technologies, including RNA-Seq. We described the changes in the global metabolism of the strain and confirmed the uniqueness of its behavior. The whole experiment demonstrated a good reproducibility. Therefore, we will be able to repeat the experiment under selected conditions in order to investigate particular metabolic changes and signaling pathways suitable for following targeted engineering.
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Project description:Acetone-butanol-ethanol (ABE) fermentation is a traditional way for solvents production through bioconversion by Clostridium species. It is still a challenge to obtain metabolic engineering strains with high ABE yield. Screening strains with remarkable characteristics from nature and improving ABE yield by mutation are viable approaches. Clostridium beijerinckii XH 0906, a newly isolated strain, produces butanol and isopropanol (BI) as the main end-products (9.1 g/L BI) during fermentation with glucose as the sole carbon source. The screening process for this strain was performed under aerobic conditions rather than anaerobic environment. Thus, it is a robust stain capable of oxygen-tolerant BI fermentation. Furthermore, C. beijerinckii XH 0906 fermented xylose and glucose simultaneously to produce BI. A mutant strain obtained by ultraviolet (UV) mutagenesis, C. beijerinckii XH 29, had improved BI production capacity and could produce 17.0 g/L BI and 18.4 g/L BI using glucose or corn stover hydrolysate, respectively as the carbon source. Interestingly, C. beijerinckii XH 29 also produced up to 19.3 g/L isopropanol through fermentation of a glucose-acetone mix. These results indicate that C. beijerinckii XH 29 is an excellent BI producer with great potential for industrial applications.
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| S-EPMC5894183 | biostudies-literature
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| S-EPMC7098912 | biostudies-literature
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.
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Project description:Purpose:C. beijerinckii DSM 6423 is the most cited natural IpOH producer. Improving the natural production of this strain through a targeted approach required a full sequencing and characterization of its genome, together with transcriptomic analyses of its own regulations.The goals of this study are then to evaluate the transcriptional profile (RNA-Seq) of C. beijerinckii DSM6423, a natural isopropanol producer, during a fermentation of glucose in controlled bioreactors. Methods : A RNA-Seq approach was chosen in order to have a timelapse study of DSM 6423 throughout the fermentation process. Three independent duplicate fermentations of DSM 6423 were carried out in bioreactors on three different weeks, showing good reproducibility. On each cultivation, five biomass samples were collected for RNA-Seq analyses.and DNA was eliminated after DNAse I treatment (AM1906, Invitrogen). The 15 resulting RNA samples were sequenced and analyzed using the previously reconstructed genome of DSM 6423. Results: Using a data analysis workflow (TAMARA) developed by the Genoscope platform of Evry, we were able to highlight the transcriptional regulation along the fermentation by calculating the transcription profiles of each gene, using the 3h sample as reference. Clustering was performed using CAST algorithm revealed 8 clusters containing 953 genes and corresponding to genes up-regulated at 6, 8, 11 or 24 hours and gene down-regulated at 6, 8, 11 or 24 hours. Conclusion : Such analyses were carried out in this study and provide useful data to better understand the genetic background and the physiological specificities of C. beijerinckii DSM6423 isopropanol producer. Notably, this work is the first omic study of a natural IBE producer. The data gathered needs time for proper exploitation, but a better understanding of the metabolic pathways and various genes involved opens the door for future targeted approaches.
2018-03-05 | GSE100024 | GEO
Project description:Clostridium saccharoperbutylacetonicum N1-4 (Csa) is a historically significant anaerobic bacterium which can perform saccharolytic fermentations to produce acetone, butanol, and ethanol (ABE). Recent genomic analyses have highlighted this organism's potential to produce polyketide and nonribosomal peptide secondary metabolites, but little is known regarding the identity and function of these metabolites. This study provides a detailed bioinformatic analysis of seven biosynthetic gene clusters (BGCs) present in the Csa genome that are predicted to produce polyketides/nonribosomal peptides. An RNA-seq-based untargeted transcriptomic approach revealed that five of seven BGCs were expressed during ABE fermentation. Additional characterization of a highly expressed nonribosomal peptide synthetase gene led to the discovery of its associated metabolite and its biosynthetic pathway. Transcriptomic analysis suggested an association of this nonribosomal peptide synthetase gene with butanol tolerance, which was supported by butanol challenge assays.
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Project description:The main bottleneck in the return of industrial butanol production from renewable feedstock through acetone-butanol-ethanol (ABE) fermentation by clostridia, such as Clostridium beijerinckii, is the low final butanol concentration. The problem is caused by the high toxicity of butanol to the production cells, and therefore, understanding the mechanisms by which clostridia react to butanol shock is of key importance. Detailed analyses of transcriptome data that were obtained after butanol shock and their comparison with data from standard ABE fermentation have resulted in new findings, while confirmed expected population responses. Although butanol shock resulted in upregulation of heat shock protein genes, their regulation is different than was assumed based on standard ABE fermentation transcriptome data. While glucose uptake, glycolysis, and acidogenesis genes were downregulated after butanol shock, solventogenesis genes were upregulated. Cyclopropanation of fatty acids and formation of plasmalogens seem to be significant processes involved in cell membrane stabilization in the presence of butanol. Surprisingly, one of the three identified Agr quorum-sensing system genes was upregulated. Upregulation of several putative butanol efflux pumps was described after butanol addition and a large putative polyketide gene cluster was found, the transcription of which seemed to depend on the concentration of butanol.
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