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Capp-Switch library preparation protocol and manual data analysis. (a) Capp-Switch and RNA-seq libraries are constructed starting from isolated bacterial RNA. For Capp-Switch libraries, primary transcripts bearing a triphosphate at the 59 end are first selectively purified using streptavidin beads. Both Capp-Switch and RNA-seq libraries were reverse transcribed using a template-switching enzyme and sequenced on the Illumina platform. (b to d) Raw Capp-Switch reads mapped on the genome of C. acetobutylicum ATCC 824. Predicted sense transcription start sites (TSSs) are shown as blue triangles, antisense TSSs are orange triangles. Predicted open reading frames are shown in yellow. (b) Opposing TSSs flank the ferredoxin gene CA_C0303. (c) Two alternative TSSs are located upstream of the pyruvate ferredoxin oxidoreductase gene CA_C2229. (d) Detection of a new coding sequence (orange) downstream from CA_P0016.
Source publication
Innovative processes to transform plant biomass into renewable chemicals are needed to replace fossil fuels and limit climate change. Clostridium acetobutylicum is of industrial interest because it ferments sugars into acetone, butanol and ethanol (ABE). However, this organism is unable to depolymerize cellulose, limiting its use for the direct tra...
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... TSS discovery in C. acetobutylicum from raw Capp-Switch data. The Capp-Switch seq library preparation pipeline (Fig. 1a) (3) was applied to RNA samples extracted from mid-log-phase C. beijerinckii DSM 6423, NCIMB 8052, and C. acetobutylicum ATCC 824 cultures growing on glucose. C. acetobutylicum raw reads sequencing data were first examined manually using Geneious software (https://www.geneious .com) to identify TSSs along the chromosome and pSOL plasmid ...
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... total of 1,250 TSSs were manually identified, of 94.8% were purines (524 A and 661 G). This TSS mapping identified novel transcriptional features (Fig. 1b to d). For example, expression of the CA_C0303 gene, encoding a ferredoxin, is flanked by inward-facing TSS (Fig. 1b), suggesting that the antisense transcript could regulate CA_C0303 expression. As a second example, transcription of CA_C2229, encoding the pyruvate ferredoxin oxidoreductase, appears to be initiated at two upstream sites ...
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... total of 1,250 TSSs were manually identified, of 94.8% were purines (524 A and 661 G). This TSS mapping identified novel transcriptional features (Fig. 1b to d). For example, expression of the CA_C0303 gene, encoding a ferredoxin, is flanked by inward-facing TSS (Fig. 1b), suggesting that the antisense transcript could regulate CA_C0303 expression. As a second example, transcription of CA_C2229, encoding the pyruvate ferredoxin oxidoreductase, appears to be initiated at two upstream sites (Fig. 1c). It can be hypothesized that alternative TSSs are the result of transcription initiated by polymerases ...
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... (Fig. 1b to d). For example, expression of the CA_C0303 gene, encoding a ferredoxin, is flanked by inward-facing TSS (Fig. 1b), suggesting that the antisense transcript could regulate CA_C0303 expression. As a second example, transcription of CA_C2229, encoding the pyruvate ferredoxin oxidoreductase, appears to be initiated at two upstream sites (Fig. 1c). It can be hypothesized that alternative TSSs are the result of transcription initiated by polymerases bound to alternative sigma factors, which might help regulate gene expression depending on specific conditions such as environmental stimuli or redox state. As a third example of how TSS data reveal novel transcript features, ...
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... the result of transcription initiated by polymerases bound to alternative sigma factors, which might help regulate gene expression depending on specific conditions such as environmental stimuli or redox state. As a third example of how TSS data reveal novel transcript features, identification of a TSS at position 17,288 in the pSOL megaplasmid (Fig. 1d) lead to the identification of an unannotated coding sequence (8,9). The product of this gene is identical to holin-like toxins identified in the Clostridium genus and shares 41.2% identity with the antibacterial protein Tmp1 ...
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... outside the associated gene, defined as having the closest gene start or end relative to the TSS). We performed RNA-seq on the same RNA samples that were used to prepare Capp-Switch seq libraries. We then normalized the strength of each TSS (in RPM) with the RNA-seq expression (in transcripts per kilobase million [TPM]) of its associated gene (Fig. S1 in the supplemental material). Because each identified TSS is associated with a normalized RPM value which is dependent on gene expression, this method allowed us to retain, in practice, an invariable, simple, normalized RPM cutoff at the end of the data analysis pipeline (10 normalized RPM, or 25 for a higher ...
Citations
... According to Fig. 4b, to the higher volume of Clac in the inoculum, the higher was the BioH 2 production. Although several anaerobic species of Clostridium can hydrolyze cellulose through the cellulosome secretion, Clac secretes very small quantities of this multi-enzyme complex, therefore, it is only considered a solventogenic strain [57]. Fig. 4c, shows that the lowest pH value (5.5) favored the production of BioH 2 (331.6 mL), thus the increase of pH from 6.5 to 7.5 decreased the gas production. ...
Bovine ruminal fluid (BRF) bioaugmented with Clostridium acetobutylicum (Clac) was assessed for hydrolyzing cellulose and produce biohydrogen (BioH2) simultaneously from pretreated corncob in a single step, without the use of external hydrolytic biocatalysts. The corncob was pretreated using three thermochemical methods: H2SO4 2%, 160 °C; NaOH 2%, 140 °C; NaOCl 2%, 140 °C; autohydrolysis: H2O, 190 °C. Subsequently, BioH2 production was carried out using the pretreated material with the highest digestibility applying a Taguchi experimental array to identify the optimal operating conditions. The results showed a higher glucose released from pretreated corncob with H2SO4 (134.7 g/L) compared to pretreated materials by autohydrolysis, NaOH and NaOCl (123 g/L, 89.8 g/L and 52.9 g/L, respectively). The mixed culture was able to hydrolyze the pretreated corncob and produce 575 mL of H2 (at 35 °C, pH 5.5, 1:2 ratio of BRF:Clac and 5% of solids loading) equivalent to 132 L H2/Kg of biomass.
... Several methods including, the chemical pretreatment, employing commercial enzyme on saccharification, co- culture of two bacterial strain, and simultaneous saccharification by Clostridium are applied to overcome the barrier for releasing fermentable sugar (Table 2). However, previous studies related to saccharification by Clostridium showed the low-solvent yield due to the weak amylase enzyme activity of Clostridia [34,35]. Additionally, in a study by Tran et al., the co-culture of Bacillus subtilis and Clostridium butylicum TISTR 1032 was reported that the highest ABE concentration was measured at 7.40 g/l from 40 g/l starch, which was a 2-fold less than the two-step fermentation [18]. ...
In Asia, uneaten cooked rice is the highest portion amongst many forms of food wastes that are thrown away. In order to make use of the thrown-away rice and potentially use it for liquid fuels, steamed Japanese rice was evaluated on biobutanol production through a two-step fermentation by amylase-producing Aspergillus oryzae, and solvent-producing Clostridium acetobutylicum YM1. The effects of sterilization and providing anaerobic conditions on solvent production in acetone-butanol-ethanol (ABE) fermentation cannot be underestimated. Several conditions, including aerobic, anaerobic, sterile, and non-sterile were investigated concerning the solvent production capability of Clostridium acetobutylicum YM1. The maximum solvent production was 11.02 ± 0.22 g/l butanol and 18.03 ± 0.34 g/l total ABE from 75 g/l dried rice. The results confirmed that the solvent production performance of the YM1 strain was not affected by the sterilization conditions. In particular, 10.91 ± 0.16 g/l butanol and 16.68 ± 0.22 g/l ABE were produced under non-sterile and aerobic conditions, which can reduce industrial-scale production costs.
In the quest for identifying novel renewable energy sources, higher alcohols from fermentative processes have received enormous interest in the last decades. Commercial microbial butanol production through the traditional acetone-butanol-ethanol process was common in the first half of the 20th century, and many attempts are underway to revive this process for butanol production. In addition to butanol, other linear and branched higher alcohols hold great promise as alternative energy sources. Although Clostridium species can naturally produce butanol, most of the other higher alcohols are synthesized in nonnative hosts. This requires the construction of novel pathways, the introduction of heterologous genes, and extensive genetic manipulation of host strains. Therefore, this chapter aims to demonstrate metabolic pathways for the synthesis of various higher alcohols. Moreover, in this chapter, metabolic engineering studies for the production of higher alcohols are reviewed. Recent advances and challenges associated with the microbial synthesis of higher alcohols are discussed.
The second-generation biobutanol production from lignocellulosic material is one of the key interests for research, considering the future of alternative energies based on the circular economy action plans. An important part of the butanol production process depends on the substrate, and therefore on the pretreatment technologies available for decreasing costs and to obtain the highest release of sugars, for a better performance of the microorganism during fermentation. This chapter is a current overview of the diverse pretreatment processes based on their classification, importance, drawbacks, and applications with the objective of facilitating to identify the elements to be considered specifically for butanol production using Clostridium strains.
