Transcriptional analysis of amino acid, metal ion, vitamin and carbohydrate uptake in butanol-producing Clostridium beijerinckii NRRL B-598

Vasylkivska, Maryna; Jureckova, Katerina; Branská, Barbora; Sedlář, Karel; Kolek, Jan; Provazník, Ivo; Patáková, Petra

doi:10.1371/journal.pone.0224560

Cited by 17 publications

(16 citation statements)

References 80 publications

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“…These findings contributed to the modification of growth media to improve either the sporulation or the solvent formation. As shown in a previous transcriptomic study on C. beijerinckii (Vasylkivska et al, 2019), the expression of genes involved in amino acids, vitamins, and ions transport varied depending on the culture's growth stage. Based on this work, close to 600 genes, putatively involved in amino acid, ions and vitamin transport and metabolism, were identified.…”

Section: Amino Acid Ions and Vitamin Transport And Metabolismmentioning

confidence: 65%

Transcriptomic and Phenotypic Analysis of a spoIIE Mutant in Clostridium beijerinckii

et al. 2020

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SpoIIE is a phosphatase involved in the activation of the first sigma factor of the forespore, σ F , during sporulation. A spoIIE mutant of Clostridium beijerinckii NCIMB 8052, previously generated by CRISPR-Cas9, did not sporulate but still produced granulose and solvents. Microscopy analysis also showed that the cells of the spoIIE mutant are elongated with the presence of multiple septa. This observation suggests that in C. beijerinckii, SpoIIE is necessary for the completion of the sporulation process, as seen in Bacillus and Clostridium acetobutylicum. Moreover, when grown in reactors, the spoIIE mutant produced higher levels of solvents than the wild type strain. The impact of the spoIIE inactivation on gene transcription was assessed by comparative transcriptome analysis at three time points (4 h, 11 h and 23 h). Approximately 5% of the genes were differentially expressed in the mutant compared to the wild type strain at all time points. Out of those only 12% were known sporulation genes. As expected, the genes belonging to the regulon of the sporulation specific transcription factors (σ F , σ E , σ G , σ K) were strongly down-regulated in the mutant. Inactivation of spoIIE also caused differential expression of genes involved in various cell processes at each time point. Moreover, at 23 h, genes involved in butanol formation and tolerance, as well as in cell motility, were up-regulated in the mutant. In contrast, several genes involved in cell wall composition, oxidative stress and amino acid transport were down-regulated. These results indicate an intricate interdependence of sporulation and stationary phase cellular events in C. beijerinckii.

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Section: Amino Acid Ions and Vitamin Transport And Metabolismmentioning

confidence: 65%

Transcriptomic and Phenotypic Analysis of a spoIIE Mutant in Clostridium beijerinckii

et al. 2020

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“…Studies of the transcriptome of wild-type and mutant C. beijerinckii cultures during fermentation indicated changes in the expression of genes involved in ion- and amino acid transport at sporulation initiation. In C. beijerinckii NRRL B598, sporulation initiation was concomitant with an upregulation of the genes encoding a magnesium transporter, and an upregulation of genes encoding potassium, sodium, and iron transporters was detected during stationary phase (Vasylkivska et al 2019 ). In cultures of the asporogenous C. beijerinckii Δ spoIIE strain, the expression of genes encoding iron transporters were downregulated during stationary phase (Diallo et al 2020b ), indicating a potential role of iron in sporulation.…”

Section: Sporulation Regulation In Solventogenic Clostridiamentioning

confidence: 99%

Sporulation in solventogenic and acetogenic clostridia

Diallo

Kengen

López-Contreras

2021

Appl Microbiol Biotechnol

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The Clostridium genus harbors compelling organisms for biotechnological production processes; while acetogenic clostridia can fix C1-compounds to produce acetate and ethanol, solventogenic clostridia can utilize a wide range of carbon sources to produce commercially valuable carboxylic acids, alcohols, and ketones by fermentation. Despite their potential, the conversion by these bacteria of carbohydrates or C1 compounds to alcohols is not cost-effective enough to result in economically viable processes. Engineering solventogenic clostridia by impairing sporulation is one of the investigated approaches to improve solvent productivity. Sporulation is a cell differentiation process triggered in bacteria in response to exposure to environmental stressors. The generated spores are metabolically inactive but resistant to harsh conditions (UV, chemicals, heat, oxygen). In Firmicutes, sporulation has been mainly studied in bacilli and pathogenic clostridia, and our knowledge of sporulation in solvent-producing or acetogenic clostridia is limited. Still, sporulation is an integral part of the cellular physiology of clostridia; thus, understanding the regulation of sporulation and its connection to solvent production may give clues to improve the performance of solventogenic clostridia. This review aims to provide an overview of the triggers, characteristics, and regulatory mechanism of sporulation in solventogenic clostridia. Those are further compared to the current knowledge on sporulation in the industrially relevant acetogenic clostridia. Finally, the potential applications of spores for process improvement are discussed.Key Points• The regulatory network governing sporulation initiation varies in solventogenic clostridia.• Media composition and cell density are the main triggers of sporulation.• Spores can be used to improve the fermentation process.

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“…Culture growth was measured as the optical density OD of the culture broth at 600 nm (Varian Cary 50 UV-Vis spectrophotometer, Agilent) against TYA medium as a blank (Vasylkivska et al, 2019).…”

Section: Methodsmentioning

confidence: 99%

“…For the first two approaches, when EMS was used as a mutagenic agent, a spore suspension of C. beijerinckii NRRL B-598 was heated to 80 • C for 2 min, vortexed and transferred to Erlenmeyer flasks with TYA medium (Vasylkivska et al, 2019) containing 20 g/L of glucose (analytical reagent grade, PENTA, Chrudim, Czechia). The strain was cultivated in a Concept 400 anaerobic chamber (Ruskinn Technology) under a stable N 2 atmosphere at 37 • C (Figure 1, step 1).…”

Section: Mutagenesismentioning

confidence: 99%

Phenotypic and Genomic Analysis of Clostridium beijerinckii NRRL B-598 Mutants With Increased Butanol Tolerance

Vasylkivska

Branská

Sedlář

et al. 2020

Front. Bioeng. Biotechnol.

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N-Butanol, a valuable solvent and potential fuel extender, can be produced via acetonebutanol-ethanol (ABE) fermentation. One of the main drawbacks of ABE fermentation is the high toxicity of butanol to producing cells, leading to cell membrane disruption, low culture viability and, consequently, low produced concentrations of butanol. The goal of this study was to obtain mutant strains of Clostridium beijerinckii NRRL B-598 with improved butanol tolerance using random chemical mutagenesis, describe changes in their phenotypes compared to the wild-type strain and reveal changes in the genome that explain improved tolerance or other phenotypic changes. Nine mutant strains with stable improved features were obtained by three different approaches and, for two of them, ethidium bromide (EB), a known substrate of efflux pumps, was used for either selection or as a mutagenic agent. It is the first utilization of this approach for the development of butanol-tolerant mutants of solventogenic clostridia, for which generally there is a lack of knowledge about butanol efflux or efflux mechanisms and their regulation. Mutant strains exhibited increase in butanol tolerance from 36% up to 127% and the greatest improvement was achieved for the strains for which EB was used as a mutagenic agent. Additionally, increased tolerance to other substrates of efflux pumps, EB and ethanol, was observed in all mutants and higher antibiotic tolerance in some of the strains. The complete genomes of mutant strains were sequenced and revealed that improved butanol tolerance can be attributed to mutations in genes encoding typical stress responses (chemotaxis, autolysis or changes in cell membrane structure), but, also, to mutations in genes X276_07980 and X276_24400, encoding efflux pump regulators. The latter observation confirms the importance of efflux in butanol stress response of the strain and offers new targets for rational strain engineering.

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Transcriptional analysis of amino acid, metal ion, vitamin and carbohydrate uptake in butanol-producing Clostridium beijerinckii NRRL B-598

Cited by 17 publications

References 80 publications

Transcriptomic and Phenotypic Analysis of a spoIIE Mutant in Clostridium beijerinckii

Transcriptomic and Phenotypic Analysis of a spoIIE Mutant in Clostridium beijerinckii

Sporulation in solventogenic and acetogenic clostridia

Phenotypic and Genomic Analysis of Clostridium beijerinckii NRRL B-598 Mutants With Increased Butanol Tolerance

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