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Promoter comparison in the three strains. (a and b) Venn diagrams showing the number of (a) InterS and (b) IntraS TSSs conserved and not conserved after ortholog pair identification, promoter alignment, and threshold-based selection. (c to f) Selected examples of TSSs with or without conservation in the three strains.
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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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... detected for genes involved in transcriptional control, redox control, and sugar uptake, suggesting that antisense transcription may regulate some of these important cellular processes in vivo. Confirmation of antisense transcription from some InterA and IntraA TSS was achieved by mapping visualization of forward reads from paired-read RNAseq (Fig. S5). The 59 UTR lengths (measured as distances in bp between InterS TSS positions and corresponding coding DNA sequence [CDS] starts) rarely exceeded 200 bp in the three strains (Fig. 3e), exhibiting no correlation with gene expression (Data Set S8). Most 59 UTRs were between 0 and 100 bp long (with a peak at 232 bp relative to the start ...
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... An alignment score threshold was chosen for each pair of strains based on the distribution of alignment scores. This pipeline allowed the recovery of 1,396 and 691 conserved InterS and IntraS promoters, respectively (Table S6). Alignments were further filtered so that each promoter was only associated with a single promoter from another strain (Fig. 5a and b). Most strikingly, the number of associated C. beijerinckii-C. acetobutylicum promoters was very low (%10% of C. acetobutylicum promoters from genes which have orthologs in C. beijerinckii were conserved in at least one of the C. beijerinckii strains), underlining a poor conservation of promoters between these species. Despite their ...
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... subsequently looked at promoter conservation at the gene level and found some interesting examples (Fig. 5c to f). Hsp18 is an important heat shock protein in solventogenic clostridia, as it is induced at the onset of solventogenesis and presumably involved in solvent resistance (19)(20)(21). The corresponding promoter is well conserved in all three strains, but the 59 UTR lengths are different (C. acetobutylicum, 149 bp, as previously reported ...
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... heat shock protein in solventogenic clostridia, as it is induced at the onset of solventogenesis and presumably involved in solvent resistance (19)(20)(21). The corresponding promoter is well conserved in all three strains, but the 59 UTR lengths are different (C. acetobutylicum, 149 bp, as previously reported [19]; C. beijerinckii, 39 bp, Fig. 5c). This could be linked to additional post-transcriptional gene regulation in C. acetobutylicum. Indeed, the 149-bp 59 UTR from C. acetobutylicum reveals an extensive secondary structure (as shown by using RNAfold with default parameters [22]), which could regulate transcript translation via a riboswitch-like mechanism, i.e., by ...
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... Because of their putative regulatory role, intragenic promoters are interesting to analyze, since strong conservation might imply a relevant functional role. In the case of the RNase Y gene, which codes for an essential protein involved in mRNA decay (25), the canonical promoter was well conserved in all three strains, with similar 59 UTR lengths (Fig. 5e). However, in both C. beijerinckii strains, very strong internal promoters were also detected. In particular, an antisense promoter strongly conserved in these two strains might repress RNase Y expression via a RNA polymerase collision mechanism (26) (supported by strand-specific RNA-seq reads; Fig. S5A). For the anti-sigma factor CsfB, ...
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... three strains, with similar 59 UTR lengths (Fig. 5e). However, in both C. beijerinckii strains, very strong internal promoters were also detected. In particular, an antisense promoter strongly conserved in these two strains might repress RNase Y expression via a RNA polymerase collision mechanism (26) (supported by strand-specific RNA-seq reads; Fig. S5A). For the anti-sigma factor CsfB, an inhibitor of the sporulation-and solventogenesis-related s G and s E factors (27), an especially strong intragenic promoter is conserved in all three strains (Fig. 5f). Hence, this promoter might be involved in the regulation of this gene in the three strains, and therefore in the regulation of ...
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... in these two strains might repress RNase Y expression via a RNA polymerase collision mechanism (26) (supported by strand-specific RNA-seq reads; Fig. S5A). For the anti-sigma factor CsfB, an inhibitor of the sporulation-and solventogenesis-related s G and s E factors (27), an especially strong intragenic promoter is conserved in all three strains (Fig. 5f). Hence, this promoter might be involved in the regulation of this gene in the three strains, and therefore in the regulation of solvent production and spore ...
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... (12), which identified secondary intragenic TSSs as a highly conserved feature in various Shewanella species. Our results, however, indicate that promoters diverged sufficiently between C. acetobutylicum and C. beijerinckii so that only a few of them were conserved. However, these promoters most likely drive crucial functions (see examples from Fig. 5). Between both C. beijerinckii strains, intergenic and intragenic promoter conservation is, as expected, much higher. Conservation of these promoters, however, does not seem to be related to gene function (Fig. S8), which is surprising given our initial hypotheses. This observation could be explained by the fact that crucial functions ...
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.
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.
