RNA-seq analysis of glucose fermentation by the natural Isopropanol producer Clostridium beijerinckii DSM6423

Purpose:C. beijerinckii DSM 6423 is the most cited natural IpOH producer. Improving the natural production of this strain through a targeted approach required a full sequencing and characterization of its genome, together with transcriptomic analyses of its own regulations.The goals of this study are then to evaluate the transcriptional profile (RNA-Seq) of C. beijerinckii DSM6423, a natural isopropanol producer, during a fermentation of glucose in controlled bioreactors. Methods : A RNA-Seq approach was chosen in order to have a timelapse study of DSM 6423 throughout the fermentation process. Three independent duplicate fermentations of DSM 6423 were carried out in bioreactors on three different weeks, showing good reproducibility. On each cultivation, five biomass samples were collected for RNA-Seq analyses.and DNA was eliminated after DNAse I treatment (AM1906, Invitrogen). The 15 resulting RNA samples were sequenced and analyzed using the previously reconstructed genome of DSM 6423. Results: Using a data analysis workflow (TAMARA) developed by the Genoscope platform of Evry, we were able to highlight the transcriptional regulation along the fermentation by calculating the transcription profiles of each gene, using the 3h sample as reference. Clustering was performed using CAST algorithm revealed 8 clusters containing 953 genes and corresponding to genes up-regulated at 6, 8, 11 or 24 hours and gene down-regulated at 6, 8, 11 or 24 hours. Conclusion : Such analyses were carried out in this study and provide useful data to better understand the genetic background and the physiological specificities of C. beijerinckii DSM6423 isopropanol producer. Notably, this work is the first omic study of a natural IBE producer. The data gathered needs time for proper exploitation, but a better understanding of the metabolic pathways and various genes involved opens the door for future targeted approaches. Overall design: Comparative analysis of samples taken from a serie a fermentations performed in controled conditions

研究背景：拜氏梭菌（C. beijerinckii）DSM 6423是被引用最多的天然异丙醇（isopropanol, IpOH）生产菌株。若要通过靶向策略提升该菌株的天然产物产量，需对其基因组进行完整测序与表征，并结合转录组分析其自身调控机制。本研究的目标为：在可控生物反应器中开展葡萄糖发酵实验时，对产异丙醇拜氏梭菌DSM 6423的转录谱（RNA测序，RNA-Seq）进行评估。
实验方法：本研究选用RNA-Seq技术，开展DSM 6423发酵过程的时间进程研究。分别于三周内开展三次独立的重复发酵实验，结果显示实验重复性良好。每次培养过程中，收集5份菌体样品用于RNA-Seq分析，经脱氧核糖核酸酶I（DNAse I，货号AM1906，Invitrogen）处理后去除基因组DNA。最终获得的15份RNA样品完成测序，并基于此前已重建的DSM 6423基因组进行数据分析。
实验结果：借助法国埃夫里根斯科普（Genoscope）平台开发的TAMARA数据分析流程，本研究以3小时采样点作为参照，通过计算每个基因的转录谱，揭示了发酵过程中的转录调控规律。采用CAST聚类算法进行聚类分析，共得到8个基因簇，涵盖953个基因，分别对应在发酵6、8、11或24小时上调表达，以及在上述时间点下调表达的基因。
研究结论：本研究开展的相关分析可为深入理解产异丙醇拜氏梭菌DSM 6423的遗传背景与生理特性提供有价值的数据支撑。值得一提的是，本工作是首个针对天然IBE（IBE）生产菌株的组学研究。收集的相关数据虽需投入时间进行充分挖掘，但对代谢通路及相关基因的深入解析，为未来开展靶向改造策略奠定了基础。
整体实验设计：对可控条件下开展的一系列发酵实验所采集的样品进行比较分析。