Effects of supplementary butyrate on butanol production and the metabolic switch in Clostridium beijerinckii NCIMB 8052: genome-wide transcriptional analysis with RNA-Seq

Biotechnology for Biofuels

Yi Wang¹  Xiangzhen Li²  Hans P Blaschek³ 

[1] Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA

[2] Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China

[3] Center for Advanced Bioenergy Research (CABER), University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA

关键词: RNA-Seq;    Transcriptional analysis;    Solventogenesis;    Metabolic switch;    Clostridium beijerinckii;    ABE fermentation;    Butanol;    Butyrate;   

Others  :  797891
DOI  :  10.1186/1754-6834-6-138

 received in 2013-07-29, accepted in 2013-09-25,  发布年份 2013

【 摘 要 】

Background

Butanol (n-butanol) has high values as a promising fuel source and chemical feedstock. Biobutanol is usually produced by the solventogenic clostridia through a typical biphasic (acidogenesis and solventogenesis phases) acetone-butanol-ethanol (ABE) fermentation process. It is well known that the acids produced in the acidogenic phase are significant and play important roles in the switch to solventogenesis. However, the mechanism that triggers the metabolic switch is still not clear.

Results

Sodium butyrate (40 mM) was supplemented into the medium for the ABE fermentation with Clostridium beijerinckii NCIMB 8052. With butyrate addition (reactor R1), solvent production was triggered early in the mid-exponential phase and completed quickly in < 50 h, while in the control (reactor R2), solventogenesis was initiated during the late exponential phase and took > 90 h to complete. Butyrate supplementation led to 31% improvement in final butanol titer, 58% improvement in sugar-based yield, and 133% improvement in butanol productivity, respectively. The butanol/acetone ratio was 2.4 versus 1.8 in the control, indicating a metabolic shift towards butanol production due to butyrate addition. Genome-wide transcriptional dynamics was investigated with RNA-Seq analysis. In reactor R1, gene expression related to solventogenesis was induced about 10 hours earlier when compared to that in reactor R2. Although the early sporulation genes were induced after the onset of solventogenesis in reactor R1 (mid-exponential phase), the sporulation events were delayed and uncoupled from the solventogenesis. In contrast, in reactor R2, sporulation genes were induced at the onset of solventogenesis, and highly expressed through the solventogenesis phase. The motility genes were generally down-regulated to lower levels prior to stationary phase in both reactors. However, in reactor R2 this took much longer and gene expression was maintained at comparatively higher levels after entering stationary phase.

Conclusions

Supplemented butyrate provided feedback inhibition to butyrate formation and may be re-assimilated through the reversed butyrate formation pathway, thus resulting in an elevated level of intracellular butyryl phosphate, which may act as a phosphate donor to Spo0A and then trigger solventogenesis and sporulation events. High-resolution genome-wide transcriptional analysis with RNA-Seq revealed detailed insights into the biochemical effects of butyrate on solventogenesis related-events at the gene regulation level.

【 授权许可】

   
2013 Wang et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Dürre P: Biobutanol: an attractive biofuel. Biotechnol J 2007, 2:1525-1534.
• [2]Jones DT, Woods DR: Acetone-butanol fermentation revisited. Microbiol Rev 1986, 50:484-524.
• [3]Terracciano JS, Kashket ER: Intracellular conditions required for initiation of solvent production by Clostridium acetobutylicum. Appl Environ Microb 1986, 52:86-91.
• [4]Husemann MHW, Papoutsakis ET: Solventogenesis in Clostridium acetobutylicum fermentation related to carboxylic acid and proton concentration. Biotechnol Bioeng 1988, 32:843-852.
• [5]Lee SM, Cho MO, Park CH, Chung YC, Kim JH, Sang BI, Um Y: Continuous butanol production using suspended and immobilized Clostridium beijerinckii NCIMB 8052 with supplementary butyrate. Energ Fuel 2008, 22:3459-3464.
• [6]Nagalakshmi U, Wang Z, Waern K, Shou C, Raha D, Gerstein M, Snyder M: The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 2008, 320:1344-1349.
• [7]Perkins TT, Kingsley RA, Fookes MC, Gardner PP, James KD, Yu L, Assefa SA, He M, Croucher NJ, Pickard DJ, et al.: A strand-specific RNA-Seq analysis of the transcriptome of the typhoid bacillus Salmonella typhi. PLoS Genet 2009, 5:e1000569.
• [8]Severin A, Woody J, Bolon Y-T, Joseph B, Diers B, Farmer A, Muehlbauer G, Nelson R, Grant D, Specht J, et al.: RNA-Seq atlas of Glycine max: a guide to the soybean transcriptome. BMC Plant Biol 2010, 10:160. BioMed Central Full Text
• [9]Wang Y, Li X, Mao Y, Blaschek H: Genome-wide dynamic transcriptional profiling in Clostridium beijerinckii NCIMB 8052 using single-nucleotide resolution RNA-Seq. BMC Genomics 2012, 13:102. BioMed Central Full Text
• [10]Wang Z, Gerstein M, Snyder M: RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 2009, 10:57-63.
• [11]van Vliet AHM: Next generation sequencing of microbial transcriptomes: challenges and opportunities. Fems Microbiol Lett 2010, 302:1-7.
• [12]Hill MO, Gauch HG: Detrended correspondence analysis: an improved ordination technique. Vegetatio 1980, 42:47-58.
• [13]Desai RP, Harris LM, Welker NE, Papoutsakis ET: Metabolic flux analysis elucidates the importance of the acid-formation pathways in regulating solvent production by Clostridium acetobutylicum. Metab Eng 1999, 1:206-213.
• [14]Lehmann D, Hönicke D, Ehrenreich A, Schmidt M, Weuster-Botz D, Bahl H, Lütke-Eversloh T: Modifying the product pattern of Clostridium acetobutylicum: physiological effects of disrupting the acetate and acetone formation pathways. Appl Microbiol Biot 2012, 94:743-754.
• [15]Wang Y, Li X, Mao Y, Blaschek H: Single-nucleotide resolution analysis of the transcriptome structure of Clostridium beijerinckii NCIMB 8052 using RNA-Seq. BMC Genomics 2011, 12:479. BioMed Central Full Text
• [16]Chen J-S: Alcohol dehydrogenase: multiplicity and relatedness in the solvent-producing clostridia. Fems Microbiol Rev 1995, 17:263-273.
• [17]Shi Z, Blaschek HP: Transcriptional analysis of Clostridium beijerinckii NCIMB 8052 and the hyper-butanol-producing mutant BA101 during the shift from acidogenesis to solventogenesis. Appl Environ Microb 2008, 74:7709-7714.
• [18]Predich M, Nair G, Smith I: Bacillus subtilis kinAspo0Fspo0AH. J Bacteriol 1992, 174:2771-2778.
• [19]Dürre P, Hollergschwandner C: Initiation of endospore formation in Clostridium acetobutylicum. Anaerobe 2004, 10:69-74.
• [20]Kenney TJ, Moran CP Jr: Organization and regulation of an operon that encodes a sporulation-essential sigma factor in Bacillus subtilis. J Bacteriol 1987, 169:3329-3339.
• [21]Alsaker KV, Papoutsakis ET: Transcriptional program of early sporulation and stationary-phase events in Clostridium acetobutylicum. J Bacteriol 2005, 187:7103-7118.
• [22]Driks A, Losick R: Compartmentalized expression of a gene under the control of sporulation transcription factor σE in Bacillus subtilis. Proc Natl Acad Sci U S A 1991, 88:9934-9938.
• [23]Kunkel B, Kroos L, Poth H, Youngman P, Losick R: Temporal and spatial control of the mother-cell regulatory gene Bacillus subtilis. Genes Dev 1989, 3:1735-1744.
• [24]Halberg R, Kroos L: Sporulation regulatory protein SpoIIID from Bacillus subtilis activates and represses transcription by both mother-cell-specific forms of RNA polymerase. J Mol Biol 1994, 243:425-436.
• [25]Lu S, Halberg R, Kroos L: Processing of the mother-cell σ factor, σK, may depend on events occurring in the forespore during Bacillus subtilis development. Proc Natl Acad Sci U S A 1990, 87:9722-9726.
• [26]Green DH, Cutting SM: Membrane topology of the Bacillus subtilis Pro-σK processing complex. J Bacteriol 2000, 182:278-285.
• [27]Weir J, Predich M, Dubnau E, Nair G, Smith I: Regulation of spo0H, a gene coding for the Bacillus subtilis σH factor. J Bacteriol 1991, 173:521-529.
• [28]Jiang M, Shao WL, Perego M, Hoch JA: Multiple histidine kinases regulate entry into stationary phase and sporulation in Bacillus subtilis. Mol Microbiol 2000, 38:535-542.
• [29]Wadhams GH, Armitage JP: Making sense of it all: bacterial chemotaxis. Nat Rev Mol Cell Biol 2004, 5:1024-1037.
• [30]Paredes CJ, Rigoutsos I, Papoutsakis ET: Transcriptional organization of the Clostridium acetobutylicum genome. Nucleic Acids Res 2004, 32:1973-1981.
• [31]Reysenbach AL, Ravenscroft N, Long S, Jones DT, Woods DR: Characterization, biosynthesis, and regulation of granulose in Clostridium acetobutylicum. Appl Environ Microb 1986, 52:185-190.
• [32]Richter H, Qureshi N, Heger S, Dien B, Cotta MA, Angenent LT: Prolonged conversion of n-butyrate to n-butanol with Clostridium saccharoperbutylacetonicum in a two-stage continuous culture with in-situ product removal. Biotechnol Bioeng 2012, 109:913-921.
• [33]Harris LM, Desai RP, Welker NE, Papoutsakis ET: Characterization of recombinant strains of the Clostridium acetobutylicum butyrate kinase inactivation mutant: need for new phenomenological models for solventogenesis and butanol inhibition? Biotechnol Bioeng 2000, 67:1-11.
• [34]Shen CR, Lan EI, Dekishima Y, Baez A, Cho KM, Liao JC: Driving forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli. Appl Environ Microb 2011, 77:2905-2915.
• [35]Alsaker KV, Spitzer TR, Papoutsakis ET: Transcriptional analysis of spo0A overexpression in Clostridium acetobutylicum and its effect on the cell’s response to butanol stress. J Bacteriol 2004, 186:1959-1971.
• [36]Ravagnani A, Jennert KCB, Steiner E, Grunberg R, Jefferies JR, Wilkinson SR, Young DI, Tidswell EC, Brown DP, Youngman P, et al.: Spo0A directly controls the switch from acid to solvent production in solvent-forming clostridia. Mol Microbiol 2000, 37:1172-1185.
• [37]Monot F, Engasser JM, Petitdemange H: Regulation of acetone butanol production in batch and continuous cultures of Clostridium acetobutylicum. Biotechnol Bioeng 1983, 25:207-216.
• [38]Zhao YS, Tomas CA, Rudolph FB, Papoutsakis ET, Bennett GN: Intracellular butyryl phosphate and acetyl phosphate concentrations in Clostridium acetobutylicum and their implications for solvent formation. Appl Environ Microb 2005, 71:530-537.
• [39]Hartmanis MGN, Gatenbeck S: Intermediary metabolism in Clostridium acetobutylicum: levels of enzymes involved in the formation of acetate and butyrate. Appl Environ Microb 1984, 47:1277-1283.
• [40]Hartmanis MG: Butyrate kinase from Clostridium acetobutylicum. J Biol Chem 1987, 262:617-621.
• [41]Annous BA, Blaschek HP: Isolation and characterization of Clostridium acetobutylicum mutants with enhanced amylolytic activity. Appl Environ Microb 1991, 57:2544-2548.
• [42]Chen CK, Blaschek HP: Effect of acetate on molecular and physiological aspects of Clostridium beijerinckii NCIMB 8052 solvent production and strain degeneration. Appl Environ Microb 1999, 65:499-505.
• [43]Qureshi N, Lolas A, Biaschek HP: Soy molasses as fermentation substrate for production of butanol using Clostridium beijerinckii BA101. J Ind Microbiol Biot 2001, 26:290-295.
• [44]Li H, Ruan J, Durbin R: Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res 2008, 18:1851-1858.
• [45]Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B: Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 2008, 5:621-628.
• [46]Gentleman R, Carey V, Bates D, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, et al.: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 2004, 5:R80. BioMed Central Full Text
• [47]Anders S, Huber W: Differential expression analysis for sequence count data. Genome Biol 2010, 11:R106. BioMed Central Full Text
• [48]Robinson MD, McCarthy DJ, Smyth GK: EdgeR: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 2010, 26:139-140.