Transcriptomic analyses of Clostridium beijerinckii NCIMB 8052 during transition from acidogenesis to solventogenesis and under butyrate supplemented conditions

Clostridium beijerinckii;Biofuel;Butanol;Transcriptomic analysis;RNA-Seq;Butyrate supplementation

Wang, Yi

关键词: Clostridium beijerinckii;    Biofuel;    Butanol;    Transcriptomic analysis;    RNA-Seq;    Butyrate supplementation;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/34440/Wang_Yi.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

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. In this study, a single-nucleotide resolution analysis of the C. beijerinckii 8052 transcriptome was conducted using high-throughput RNA-Seq technology. The transcription start sites and operon structure throughout the genome were identified. The structure of important gene operons involved in metabolic pathways for acid and solvent production in C. beijerinckii 8052 were confirmed, including pta-ack, ptb-buk, hbd-etfA-etfB-crt (bcs) and ald-ctfA-ctfB-adc (sol) operons; important operons related to chemotaxis/motility, transcriptional regulation, stress response and fatty acids biosynthesis along with others were also defined. In addition, 20 previously non-annotated regions were discovered to be with significant transcriptional activities and 15 genes whose translation start codons were found to be likely mis-annotated. As a consequence, the accuracy of existing C. beijerinckii genome annotation was significantly enhanced. The genome-wide transcriptional dynamics of C. beijerinckii 8052 over a batch fermentation process was revealed in detail based on the RNA-Seq data. The gene expression profiles indicated that the glycolysis genes were highly expressed throughout the fermentation, with comparatively more active expression during acidogenesis phase. The expression of acid formation genes was down-regulated at the onset of solvent formation, in accordance with the metabolic pathway shift from acidogenesis to solventogenesis. The sol operon genes, were highly-coordinately expressed and up-regulated at the onset of solventogenesis. Out of the 20 genes encoding alcohol dehydrogenase in C. beijerinckii, Cbei_1722 and Cbei_2181 were highly up-regulated at the initiation of solvent production, corresponding to their key roles in primary alcohol production. Most sporulation genes in C. beijerinckii 8052 demonstrated similar temporal expression patterns to those observed in B. subtilis and C. acetobutylicum, while sporulation sigma factor genes sigE and sigG exhibited accelerated and stronger expression in C. beijerinckii 8052, which is consistent with the more rapid forespore and endspore development in this strain. Global expression patterns for specific gene functional classes demonstrated general expression profiles corresponding to the cell physiological variation and metabolic pathway switch during the fermentation. Butyrate has long been suggested as a potential triggering factor for the solventogenesis switch during the ABE fermentation. In this study, sodium butyrate was added to the chemically defined MP2 medium for ABE fermentation with C. beijerinckii 8052 in order to investigate the effects of butyrate on solvent production and metabolic pathway switch. The results indicated that with butyrate supplementation, the solvent production was triggered early in the mid-exponential phase and finished quickly in < 50 h from the time of innoculation. While in the control, solventogenesis initiated during late exponential phase and took > 90 h to reach the maximum butanol level. Butyrate supplementation led to a 31% improvement in the final butanol titer, 58% improvement in butanol yield and 133% improvement in butanol productivity, comparing to the control without butyrate addition. It also led to a higher butanol/acetone ratio compared to the control, indicating a metabolism shift towards butanol production. The addition of butyrate also triggered much earlier and stronger sporulation during the fermentation. Genome-wide transcriptional analysis was performed with RNA-Seq over the course of fermentation with and without added butyrate. With butyrate addition, the gene expression in central metabolic pathway related to solventogenesis was induced about 10 hours earlier and accelerated, and the maximum expression levels of butyryl-CoA and solvent formation genes were elevated comparing to the control, correlating with the faster and higher solvent production. In both conditions, the sporulation genes were induced at the onset of solventogenesis, although this occurred much earlier in the reactor with butyrate addition, and went through a similar temporal expression patterns with much faster down-regulation under the condition with butyrate addition. The motility related genes were generally down-regulated to lower levels just prior to the stationary phase under both conditions. However, in the control it took much longer and the gene expression remained at comparatively higher levels after the microbe entered stationary phase. Supplemented butyrate may cause feedback inhibition to butyrate formation and be re-assimilated through the reversed butyrate formation pathway, and thus result in an elevated level of intracellular butyryl phosphate, which may act as a phosphate donor to Spo0A and consequently triggered solventogenesis and related events.The results from this work provided insights for further C. beijerinckii strain improvement employing system biology-based strategies and metabolic engineering approaches. Furthermore, this work is also an essential methodology reference for conducting transcriptional analysis employing next-generation sequencing technology.