Comparative phenotypic analysis and genome sequence of Clostridium beijerinckii SA-1, an offspring of NCIMB 8052 (original) (raw)
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Scientific Reports
Clostridium beijerinckii NRRL B-598 is a sporulating, butanol and hydrogen producing strain that utilizes carbohydrates by the acetone-butanol-ethanol (ABe) fermentative pathway. the pathway consists of two metabolic phases, acidogenesis and solventogenesis, from which the latter one can be coupled with sporulation. Thorough transcriptomic profiling during a complete life cycle and both metabolic phases completed with flow cytometry, microscopy and a metabolites analysis helped to find out key genes involved in particular cellular events. the description of genes/operons that are closely involved in metabolism or the cell cycle is a necessary condition for metabolic engineering of the strain and will be valuable for all C. beijerinckii strains and other Clostridial species. the study focused on glucose transport and catabolism, hydrogen formation, metabolic stress response, binary fission, motility/ chemotaxis and sporulation, which resulted in the composition of the unique image reflecting clostridial population changes. surprisingly, the main change in expression of individual genes was coupled with the sporulation start and not with the transition from acidogenic to solventogenic metabolism. As expected, solvents formation started at pH decrease and the accumulation of butyric and acetic acids in the cultivation medium. Strictly anaerobic bacteria represent a less well known and studied group compared to their aerobic or facultatively anaerobic counterparts. Nevertheless, the present need to identify different solutions to problems threatening the ecological and energetic stability of the world has focused attention on these bacteria and has initiated an era of study revealing their powerful metabolic potential. Clostridia, a diverse group of strictly anaerobic bacteria, include known pathogenic and toxinogenic bacteria such as Clostridium difficile or Clostridium botulinum but also non-pathogenic industrially important species such as Clostridium acetobutylicum, Clostridium beijerinckii or Clostridium ljungdahlii. Among them, butanol-producers, C. acetobutylicum, C. beijerinckii, C. saccharoperbutylacetonicum and others offer a wide range of options related to substrate choice and utilization because they can produce a spectrum of hydrolytic enzymes and can utilize different, often unusual, and insufficiently described metabolic pathways to produce valuable chemical compounds that are currently produced from oil or its derivatives. Current research in the field is focused not only on strain improvement and the use of alternative, waste stream-based substrates but also on acquiring a deeper understanding of clostridial metabolism and life cycle changes. Despite the 100th year anniversary of industrial acetone-butanol-ethanol (ABE) in 2016 1 , butanol producers still managed to surprise us; for recent news in the field, see Herman et al. (2017); Jones et al. (2018); Sandoval-Espinola et al. 2-4. The genome of C. beijerinckii NRRL B-598 was assembled in 2015 5 having an original species name Clostridium pasteurianum. The strain does not contain plasmids, can produce spores, excels in oxygen tolerance and overall fitness, cannot produce isopropanol, and produces very low concentrations of ethanol but high levels
Journal of Bacteriology, 2001
The genome sequence of the solvent-producing bacterium Clostridium acetobutylicum ATCC 824 has been determined by the shotgun approach. The genome consists of a 3.94-Mb chromosome and a 192-kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison of C. acetobutylicum to Bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria. However, the C. acetobutylicum genome also contains a significant number of predicted operons that are shared with distantly related bacteria and archaea but not with B. subtilis. Phylogenetic analysis is compatible with the dissemination of such operons by horizontal transfer. The enzymes of the solventogenesis pathway and of the cellulosome of C. acetobutylicum comprise a new set of metabolic capacities not previously represented in the collection of complete genomes. These enzymes show a complex pattern of evolutionary affinities, emphasizing the role of lateral gene exchange in the evolution of the unique metabolic profile of the bacterium. Many of the sporulation genes identified in B. subtilis are missing in C. acetobutylicum, which suggests major differences in the sporulation process. Thus, comparative analysis reveals both significant conservation of the genome organization and pronounced differences in many systems that reflect unique adaptive strategies of the two gram-positive bacteria.
Assembly and Automated Annotation of the Clostridium scatologenes Genome
2012
Clostridium scatologenes is an anaerobic bacterium that demonstrates some unusual metabolic traits such as the production of 3-methyl indole. The availability of genome level sequencing has lent itself to the exploration and elucidation of unique metabolic pathways in other organisms such as Clostridium botulinum. The Clostridium scatologenes genome, with an estimated length 4.2 million bp, was sequenced by the Applied Biosystems Solid method and the Roche 454 pyrosequencing method. The resulting DNA sequences were combined and assembled into 8267 contigs with an average length of 1250 bp with the Newbler Assembler program. Comparision of published subunits of csd gene and assembled contigs identified that one contig contained all three subunits. In addition a gene with similarity to clostridium carboxidivorans butyrate kinase was found lined next to csd gene. An alignment of the contig and csd gene sequences identified three deletions in the contig within the 4066 bases of the alignment. This implies that there is about 0.07% error rate in the sequencing itself requiring more finishing. Even without finishing the genome assembly into single contig, contigs were annotated in RAST pipeline predicting 2521 protein encoding genes (PEGs). The PEGs were classified by their metabolic function and compared to classified PEGs found in the closely related clostridium species, Clostridium carboxidivorans and Clostridium. ljungdahlii, which have similarly sized genomes. According to the RAST analysis, vii Clostridium scatologenes had 35% subsystem coverage of all known metabolic processes with its 2521 PEGs. This compares to 41% for Clostridium carboxidivorans with 4174 PEGs (29) and 42% for Clostridium ljungdahlii with 4184 PEGs (30), indicating that Clostridium scatologenes may still have more genes to be identified. Comparison of the percent genes found in the metabolic subsystems was similar except in motility and chemotaxis. The contigs, on which the csd gene and tryptophan metabolizing genes lay, were examined to see if additional genes might support these metabolic pathways. Butyrate kinase was associated with the csd genes but no other associations were found for the two tryptophan metabolizing genes. The tryptophan biosynthesis operon genes were all found on one contig (contig 6771) and were syntenic with other bacterial species.
Applied and environmental microbiology, 2011
Recent advances in systems biology, omics, and computational studies allow us to carry out data mining for improving biofuel production bioprocesses. Of particular interest are bioprocesses that center on microbial capabilities to biotransform both the hexose and pentose fractions present in crop residues. This called for a systematic exploration of the components of the media to obtain higher-density cultures and more-productive fermentation operations than are currently found. By using a meta-analysis approach of the transcriptional responses to butanol stress, we identified the nutritional requirements of solvent-tolerant strain Clostridium beijerinckii SA-1 (ATCC 35702). The nutritional requirements identified were later validated using the chemostat pulse-and-shift technique. C. beijerinckii SA-1 was cultivated in a two-stage single-feed-stream continuous production system to test the proposed validated medium formulation, and the coutilization of D-glucose and D-xylose was eva...
Applied and Environmental Microbiology, 2008
Clostridium beijerinckii is an anaerobic bacterium used for the fermentative production of acetone and butanol. The recent availability of genomic sequence information for C. beijerinckii NCIMB 8052 has allowed for an examination of gene expression during the shift from acidogenesis to solventogenesis over the time course of a batch fermentation using a ca. 500-gene set DNA microarray. The microarray was constructed using a collection of genes which are orthologs of members of gene families previously found to be important to the physiology of C. acetobutylicum ATCC 824. Similar to the onset of solventogenesis in C. acetobutylicum 824, the onset of solventogenesis in C. beijerinckii 8052 was concurrent with the initiation of sporulation. However, forespores and endospores developed more rapidly in C. beijerinckii 8052 than in C. acetobutylicum 824, consistent with the accelerated expression of the sigE - and sigG -regulated genes in C. beijerinckii 8052. The comparison of gene expre...
FEMS Microbiology Reviews, 1995
A physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052 chromosome has been constructed, encompassing about 90 rare restriction sites. The 14 rrn operons together with about 40 genes have been assigned positions on the map. Genetic analysis and gene transfer have been developed in this organism to enable in vivo analysis of the roles of cloned genes using marker replacement technology. Experiments using the available genetic tools have shown that spoOA plays a cardinal role in controlling several aspects of the transition from exponential growth to stationary phase in C. beijerinckii. These include initiation of sporulation, accumulation of the storage polysaccharide, granulose, and production of acetone and butanol. Several C. beijerinckii and C. acetobutylicum genes concerned with fermentative metabolism, whose expression is modulated at the onset of solventogenesis, contain sequence motifs resembling 0A boxes in their 5' regulatory regions. This invites the speculation that they are under the direct control of Spo0A, and additional data are now required to test this prediction.
2012
Today, the exhaustion of fossil fuel resources and the deterioration of the natural environment drive people to seek alternative bio-based fuels and chemicals from renewable sources. Biobutanol produced through microbial fermentation of biomass has been of great interest because of its various advantages as a biofuel and considerable value as an industrial chemical feedstock. Clostridium beijerinckii is among the prominent species for biobutanol production as it demonstrates a broad substrate range for growth and solvent production. Although the transcriptome structure and transcriptional profiling are essential for understanding the functional and regulatory network of the genome and specific gene functions and regulations associated with the cell physiology, the physical structure of the transcriptome and the transcriptional profiles were not well understood for C. beijerinckii.
Biotechnology for Biofuels
Background: Clostridium saccharobutylicum NCP 262 is a solventogenic bacterium that has been used for the industrial production of acetone, butanol, and ethanol. The lack of a genetic manipulation system for C. saccharobutylicum currently limits (i) the use of metabolic pathway engineering to improve the yield, titer, and productivity of n-butanol production by this microorganism, and (ii) functional genomics studies to better understand its physiology. Results: In this study, a marker-less deletion system was developed for C. saccharobutylicum using the codBA operon genes from Clostridium ljungdahlii as a counterselection marker. The codB gene encodes a cytosine permease, while codA encodes a cytosine deaminase that converts 5-fluorocytosine to 5-fluorouracil, which is toxic to the cell. To introduce a marker-less genomic modification, we constructed a suicide vector containing: the catP gene for thiamphenicol resistance; the codBA operon genes for counterselection; fused DNA segments both upstream and downstream of the chromosomal deletion target. This vector was introduced into C. saccharobutylicum by tri-parental conjugation. Single crossover integrants are selected on plates supplemented with thiamphenicol and colistin, and, subsequently, double-crossover mutants whose targeted chromosomal sequence has been deleted were identified by counterselection on plates containing 5-fluorocytosine. Using this marker-less deletion system, we constructed the restrictiondeficient mutant C. saccharobutylicum ΔhsdR1ΔhsdR2ΔhsdR3, which we named C. saccharobutylicum Ch2. This triple mutant exhibits high transformation efficiency with unmethylated DNA. To demonstrate its applicability to metabolic engineering, the method was first used to delete the xylB gene to study its role in xylose and arabinose metabolism. Furthermore, we also deleted the ptb and buk genes to create a butyrate metabolism-negative mutant of C. saccharobutylicum that produces n-butanol at high yield. Conclusions: The plasmid vectors and the method introduced here, together with the restriction-deficient strains described in this work, for the first time, allow for efficient marker-less genomic modification of C. saccharobutylicum and, therefore, represent valuable tools for the genetic and metabolic engineering of this industrially important solvent-producing organism.
Biotechnology for biofuels, 2015
Biodiesel production results in crude glycerol waste from the transesterification of fatty acids (10 % w/w). The solventogenic Clostridium pasteurianum, an anaerobic Firmicute, can produce butanol from glycerol as the sole carbon source. Coupling butanol fermentation with biodiesel production can improve the overall economic viability of biofuels. However, crude glycerol contains growth-inhibiting byproducts which reduce feedstock consumption and solvent production. To obtain a strain with improved characteristics, a random mutagenesis and directed evolution selection technique was used. A wild-type C. pasteurianum (ATCC 6013) culture was chemically mutagenized, and the resulting population underwent 10 days of selection in increasing concentrations of crude glycerol (80-150 g/L). The best-performing mutant (M150B) showed a 91 % increase in butanol production in 100 g/L crude glycerol compared to the wild-type strain, as well as increased growth rate, a higher final optical density,...