Phenotypic and genomic analysis of isopropanol and 1,3-propanediol producer Clostridium diolis DSM 15410

Clostridium diolis DSM 15410 is a type strain of solventogenic clostridium capable of conducting isopropanol-butanol-ethanol fermentation. By studying its growth on different carbohydrates, we verified its ability to utilize glycerol and produce 1,3-propanediol and discovered its ability to produced isopropanol. Complete genome sequencing showed that its genome is a single circular chromosome and belongs to the cluster I (sensu scricto) of the genus Clostridium. By cultivation analysis we highlighted its specific behavior in comparison to two selected closely related strains. Despite the fact that several CRISPR loci were found, 16 putative prophages showed the ability to receive foreign DNA. Thus, the strain has the necessary features for future engineering of its 1,3-propanediol biosynthetic pathway and for the possible industrial utilization in the production of biofuels

Transcription profiling of butanol producer Clostridium beijerinckii NRRL B-598 using RNA-Seq

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

Identification and validation of reference genes in Clostridium beijerinckii NRRL B-598 for RT-qPCR using RNA-Seq data

Gene expression analysis through reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) depends on correct data normalization by reference genes with stable expression. Although Clostridium beijerinckii NRRL B-598 is a promising Gram-positive bacterium for the industrial production of biobutanol, validated reference genes have not yet been reported. In this study, we selected 160 genes with stable expression based on an RNA sequencing (RNA-Seq) data analysis, and among them, seven genes (zmp, rpoB1, rsmB, greA, rpoB2, topB2, and rimO) were selected for experimental validation by RT-qPCR and gene ontology enrichment analysis. According to statistical analyses, zmp and greA were the most stable and suitable reference genes for RT-qPCR normalization. Furthermore, our methodology can be useful for selection of the reference genes in other strains of Clostridium beijerinckii and it also suggests that the RNA-Seq data can be used for the initial selection of novel reference genes, however their validation is required

A transcriptional response of Clostridium beijerinckii NRRL B-598 to a butanol shock

Background One of the main obstacles preventing solventogenic clostridia from achieving higher yields in biofuel production is the toxicity of produced solvents. Unfortunately, regulatory mechanisms responsible for the shock response are poorly described on the transcriptomic level. Although the strain Clostridium beijerinckii NRRL B-598, a promising butanol producer, has been studied under different conditions in the past, its transcriptional response to a shock caused by butanol in the cultivation medium remains unknown. Results In this paper, we present a transcriptional response of the strain during a butanol challenge, caused by the addition of butanol to the cultivation medium at the very end of the acidogenic phase, using RNA-Seq. We resequenced and reassembled the genome sequence of the strain and prepared novel genome and gene ontology annotation to provide the most accurate results. When compared to samples under standard cultivation conditions, samples gathered during butanol shock represented a well-distinguished group. Using reference samples gathered directly before the addition of butanol, we identified genes that were differentially expressed in butanol challenge samples. We determined clusters of 293 down-regulated and 301 up-regulated genes whose expression was affected by the cultivation conditions. Enriched term “RNA binding” among down-regulated genes corresponded to the downturn of translation and the cluster contained a group of small acid-soluble spore proteins. This explained phenotype of the culture that had not sporulated. On the other hand, up-regulated genes were characterized by the term “protein binding” which corresponded to activation of heat-shock proteins that were identified within this cluster. Conclusions We provided an overall transcriptional response of the strain C. beijerinckii NRRL B-598 to butanol shock, supplemented by auxiliary technologies, including high-pressure liquid chromatography and flow cytometry, to capture the corresponding phenotypic response. We identified genes whose regulation was affected by the addition of butanol to the cultivation medium and inferred related molecular functions that were significantly influenced. Additionally, using high-quality genome assembly and custom-made gene ontology annotation, we demonstrated that this settled terminology, widely used for the analysis of model organisms, could also be applied to non-model organisms and for research in the field of biofuels

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

In-depth knowledge of cell metabolism and nutrient uptake mechanisms can lead to the development of a tool for improving acetone-butanol-ethanol (ABE) fermentation performance and help to overcome bottlenecks in the process, such as the high cost of substrates and low production rates. Over 300 genes potentially encoding transport of amino acids, metal ions, vitamins and carbohydrates were identified in the genome of the butanol-producing strain Clostridium beijerinckii NRRL B-598, based on similarity searches in protein function databases. Transcriptomic data of the genes were obtained during ABE fermentation by RNA-Seq experiments and covered acidogenesis, solventogenesis and sporulation. The physiological roles of the selected 81 actively expressed transport genes were established on the basis of their expression profiles at particular stages of ABE fermentation. This article describes how genes encoding the uptake of glucose, iron, riboflavin, glutamine, methionine and other nutrients take part in growth, production and stress responses of C. beijerinckii NRRL B-598. These data increase our knowledge of transport mechanisms in solventogenic Clostridium and may be used in the selection of individual genes for further research

Deeper below the surface – transcriptional changes of selected Clostridium beijerinckii NRRL B-598 genes induced by butanol shock

The main bottleneck in the return of industrial butanol production from renewable feedstock, by acetone-butanol-ethanol (ABE) fermentation by clostridia, such as Clostridium beijerinckii, is the low final butanol concentration. The problem is caused by 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 obtained after butanol shock, and their comparison with data from standard ABE fermentations resulted in new findings while they confirmed expected population responses as well. Although butanol shock resulted in upregulation of heat shock protein genes, their regulation is probably 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 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 be dependent on the concentration of butanol

Additional file 3: of Transcription profiling of butanol producer Clostridium beijerinckii NRRL B-598 using RNA-Seq

Putative active genes misidentified as pseudogenes due to assembly errors. (PDF 210 kb