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

Department of Biomedical Engineering Brno University of Technology Brno Czech Republic

Department of Biotechnology University of Chemistry and Technology Prague Prague Czech Republic

Zobrazit více v PubMed

Alsaker, K. V. , Paredes, C. , & Papoutsakis, E. T. (2010). Metabolite stress and tolerance in the production of biofuels and chemicals: Gene‐expression‐based systems analysis of butanol, butyrate, and acetate stresses in the anaerobe Clostridium acetobutylicum . Biotechnology and Bioengineering, 105, 1131–1147. 10.1002/bit.22628 PubMed DOI

Baerends, R. J. S. , Smits, W. K. , de Jong, A. , Hamoen, L. W. , Kok, J. , & Kuipers, O. P. (2004). Genome2D: A visualization tool for the rapid analysis of bacterial transcriptome data. Genome Biology, 5(5), R37 10.1186/gb-2004-5-5-r37 PubMed DOI PMC

Baumann, N. A. , Hagen, P. O. , & Goldfine, H. (1965). Phospholipids of Clostridium butyricum. Studies on plasmalogen composition and biosynthesis. The Journal of Biological Chemistry, 240, 1559–1567. PubMed

Behnken, S. , & Hertweck, C. (2012). Cryptic polyketide synthase genes in non‐pathogenic Clostridium spp. PLoS One, 7(1), e29609 10.1371/journal.pone.0029609 PubMed DOI PMC

Branska, B. , Fortova, L. , Dvorakova, M. , Liu, H. , Patakova, P. , Zhang, J. , & Melzoch, M. (2020). Chicken feather and wheat straw hydrolysate for direct utilization in biobutanol production. Renewable Energy, 145, 1941–1948. 10.1016/j.renene.2019.07.094 DOI

Branska, B. , Pechacova, Z. , Kolek, J. , Vasylkivska, M. , & Patakova, P. (2018). Flow cytometry analysis of Clostridium beijerinckii NRRL B‐598 populations exhibiting different phenotypes induced by changes in cultivation conditions. Biotechnology for Biofuels, 11(1), 99 10.1186/s13068-018-1096-x PubMed DOI PMC

Chang, Y. , & Cronan, J. E. (1999). Membrane cyclopropane fatty acid content is a major factor in acid resistance of Escherichia coli . Molecular Microbiology, 33(2), 249–259. 10.1046/j.1365-2958.1999.01456.x PubMed DOI

Cruz‐Morales, P. , Orellana, C. A. , Moutafis, G. , Moonen, G. , Rincon, G. , Nielsen, L. K. , & Marcellin, E. (2019). Revisiting the evolution and taxonomy of Clostridia, a phylogenomic update. Genome Biology and Evolution, 11(7), 2035–2044. 10.1093/gbe/evz096 PubMed DOI PMC

De Mendoza, D. , Schujman, G. E. , & Aguilar, P. S. (2002). Biosynthesis and function of membrane lipids In Sonenshein A., Losick R., & Hoch J. (Eds.), Bacillus subtilis and its closest relatives (pp. 43–55). American Society of Microbiology.

Deng, W. , Li, C. , & Xie, J. (2013). The underling mechanism of bacterial TetR/AcrR family transcriptional repressors. Cellular Signalling, 25(7), 1608–1613. 10.1016/j.cellsig.2013.04.003 PubMed DOI

Ezeji, T. , Qureshi, N. , & Blaschek, H. P. (2007). Butanol production from agricultural residues: Impact of degradation products on Clostridium beijerinckii growth and butanol fermentation. Biotechnology and Bioengineering, 97(6), 1460–1469. 10.1002/bit.21373 PubMed DOI

Fisher, M. A. , Boyarskiy, S. , Yamada, M. R. , Kong, N. , Bauer, S. , & Tullman‐Ercek, D. (2014). Enhancing tolerance to short‐chain alcohols by engineering the Escherichia coli AcrB efflux pump to secrete the non‐native substrate n‐butanol. ACS Synthetic Biology, 3, 30–40. 10.1021/sb400065q PubMed DOI

Goldfine, H. (2010). The appearance, disappearance and reappearance of plasmalogens in evolution. Progress in Lipid Research, 49(4), 493–498. 10.1016/j.plipres.2010.07.003 PubMed DOI

Goldfine, H. , & Johnston, N. (2005). Membrane lipids of Clostridia In Dürre P. (Ed.), Handbook on Clostridia (pp. 297–309). CRC Press.

Goodson, J. R. , Klupt, S. , Zhang, C. , Straight, P. , & Winkler, W. C. (2017). LoaP is a broadly conserved antiterminator protein that regulates antibiotic gene clusters in Bacillus amyloliquefaciens . Nature Microbiology, 2, 17003 10.1038/nmicrobiol.2017.3 PubMed DOI PMC

Han, X. , & Gross, R. W. (1990). Plasmenylcholine and phosphatidylcholine membrane bilayers possess distinct conformational motifs. Biochemistry, 29(20), 4992–4996. 10.1021/bi00472a032 PubMed DOI

Herman, N. A. , Kim, S. J. , Li, J. S. , Cai, W. , Koshino, H. , & Zhang, W. (2017). The industrial anaerobe Clostridium acetobutylicum uses polyketides to regulate cellular differentiation. Nature Communications, 8, 1514 10.1038/s41467-017-01809-5 PubMed DOI PMC

Ingram, L. O. , & Buttke, T. M. (1985). Effects of alcohols on microorganisms. Advances in Microbial Physiology, 25, 253–300. 10.1016/S0065-2911(08)60294-5 PubMed DOI

Janssen, H. , Grimmler, C. , Ehrenreich, A. , Bahl, H. , & Fischer, R. J. (2012). A transcriptional study of acidogenic chemostat cells of Clostridium acetobutylicum‐Solvent stress caused by a transient n‐butanol pulse. Journal of Biotechnology, 161(3), 354–365. 10.1016/j.jbiotec.2012.03.027 PubMed DOI

Johnston, N. C. , Goldfine, H. , Malthaner, M. , & Seelig, J. (1987). 2H‐NMR studies on ether lipid‐rich bacterial membranes: Deuterium order profile of Clostridium butyricum . BBA ‐ Biomembranes, 899(2), 302–306. 10.1016/0005-2736(87)90412-3 PubMed DOI

Kim, H. J. , Hwang, N. R. , & Lee, K. J. (2007). Heat shock responses for understanding diseases of protein denaturation. Molecules and Cells, 23(2), 123–131. PubMed

Koga, Y. , & Goldfine, H. (1984). Biosynthesis of phospholipids in Clostridium butyricum: Kinetics of synthesis of plasmalogens and the glycerol acetal of ethanolamine plasmalogen. Journal of Bacteriology, 159(2), 597–604. PubMed PMC

Kolek, J. , Patakova, P. , Melzoch, K. , Sigler, K. , & Rezanka, T. (2015). Changes in membrane plasmalogens of Clostridium pasteurianum during butanol fermentation as determined by lipidomic analysis. PLoS One, 10(3), e0122058 10.1371/journal.pone.0122058 PubMed DOI PMC

Lee, J. , & Blaschek, H. P. (2001). Glucose uptake in Clostridium beijerinckii NCIMB 8052 and the solvent‐hyperproducing mutant BA101. Applied and Environmental Microbiology, 67(11), 5025–5031. PubMed PMC

Lee, J. , Mitchell, W. J. , Tangney, M. , & Blaschek, H. P. (2005). Evidence for the presence of an alternative glucose transport system in Clostridium beijerinckii NCIMB 8052 and the solvent‐hyperproducing mutant BA101. Applied and Environmental Microbiology, 71(6), 3384–3387. 10.1128/AEM.71.6.3384-3387.2005 PubMed DOI PMC

Lee, S. Y. , Park, J. H. , Jang, S. H. , Nielsen, L. K. , Kim, J. , & Jung, K. S. (2008). Fermentative butanol production by clostridia. Biotechnology and Bioengineering, 101(2), 209–228. 10.1002/bit.22003 PubMed DOI

Lepage, C. , Fayolle, F. , Hermann, M. , & Vandecasteele, J. P. (1987). Changes in membrane lipid composition of Clostridium acetobutylicum during acetone‐butanol fermentation: Effects of solvents, growth temperature and pH. Microbiology, 133(1), 103–110. 10.1099/00221287-133-1-103 DOI

Letzel, A. C. , Pidot, S. J. , & Hertweck, C. (2013). A genomic approach to the cryptic secondary metabolome of the anaerobic world. Natural Product Reports, 30(3), 392–428. 10.1039/c2np20103h PubMed DOI

Liao, Y. , Smyth, G. K. , & Shi, W. (2014). FeatureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics, 30(7), 923–930. 10.1093/bioinformatics/btt656 PubMed DOI

Liao, Z. , Zhang, Y. , Luo, S. , Suo, Y. , Zhang, S. , & Wang, J. (2017). Improving cellular robustness and butanol titers of Clostridium acetobutylicum ATCC824 by introducing heat shock proteins from an extremophilic bacterium. Journal of Biotechnology, 252, 1–10. 10.1016/j.jbiotec.2017.04.031 PubMed DOI

Love, M. I. , Huber, W. , & Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA‐seq data with DESeq2. Genome Biology, 15(12), 550 10.1186/s13059-014-0550-8 PubMed DOI PMC

MacDonald, D. L. , & Goldfine, H. (1991). Effects of solvents and alcohols on the polar lipid composition of Clostridium butyricum under conditions of controlled lipid chain composition. Applied and Environmental Microbiology, 57(12), 3517–3521. PubMed PMC

Maiti, S. , Sarma, S. J. , Brar, S. K. , Le Bihan, Y. , Drogui, P. , Buelna, G. , & Verma, M. (2016). Agro‐industrial wastes as feedstock for sustainable bio‐production of butanol by Clostridium beijerinckii . Food and Bioproducts Processing, 98, 217–226. 10.1016/j.fbp.2016.01.002 DOI

Mann, M. S. , Dragovic, Z. , Schirrmacher, G. , & Lutke‐Eversloh, T. (2012). Over‐expression of stress protein‐encoding genes helps Clostridium acetobutylicum to rapidly adapt to butanol stress. Biotechnology Letters, 34(9), 1643–1649. 10.1007/s10529-012-0951-2 PubMed DOI

Martin, W. F. , & Sousa, F. L. (2016). Early microbial evolution: The age of anaerobes. Cold Spring Harbor Perspectives in Biology, 8(2), a018127 10.1101/cshperspect.a018127 PubMed DOI PMC

Moon, H. G. , Jang, Y. S. , Cho, C. , Lee, J. , Binkley, R. , & Lee, S. Y. (2016). One hundred years of clostridial butanol fermentation. FEMS Microbiology Letters, 363(3), fnw001 10.1093/femsle/fnw001 PubMed DOI

Mukhopadhyay, A. (2015). Tolerance engineering in bacteria for the production of advanced biofuels and chemicals. Trends in Microbiology, 23(8), 498–508. 10.1016/j.tim.2015.04.008 PubMed DOI

Murarka, P. , Bagga, T. , Singh, P. , Rangra, S. , & Srivastava, P. (2019). Isolation and identification of a TetR family protein that regulates the biodesulfurization operon. AMB Express, 9(1), 71 10.1186/s13568-019-0801-x PubMed DOI PMC

Nguyen Le Minh, P. , de Cima, S. , Bervoets, I. , Maes, D. , Rubio, V. , & Charlier, D. (2015). Ligand binding specificity of RutR, a member of the TetR family of transcription regulators in Escherichia coli . FEBS Open Bio, 5, 76–84. 10.1016/j.fob.2015.01.002 PubMed DOI PMC

Patakova, P. , Branska, B. , Sedlar, K. , Vasylkivska, M. , Jureckova, K. , Kolek, J. , Koscova, P. , & Provaznik, I. (2019). Acidogenesis, solventogenesis, metabolic stress response and life cycle changes in Clostridium beijerinckii NRRL B‐598 at the transcriptomic level. Scientific Reports, 9(1), 1371 10.1038/s41598-018-37679-0 PubMed DOI PMC

Patakova, P. , Kolek, J. , Sedlar, K. , Koscova, P. , Branska, B. , Kupkova, K. , Paulova, L. , & Provaznik, I. (2018). Comparative analysis of high butanol tolerance and production in clostridia. Biotechnology Advances, 36(3), 721–738. 10.1016/j.biotechadv.2017.12.004 PubMed DOI

Pini, C. , Bernal, P. , Godoy, P. , Ramos, J. , & Segura, A. (2009). Cyclopropane fatty acids are involved in organic solvent tolerance but not in acid stress resistance in Pseudomonas putida DOT‐T1E. Microbial Biotechnology, 2(2), 253–261. 10.1111/j.1751-7915.2009.00084.x PubMed DOI PMC

Robinson, M. D. , McCarthy, D. J. , & Smyth, G. K. (2010). EdgeR: A Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics, 26(1), 139–140. 10.1093/bioinformatics/btp616 PubMed DOI PMC

Sardessai, Y. , & Bhosle, S. (2002). Tolerance of bacteria to organic solvents. Research in Microbiology, 153(5), 263–268. 10.1016/S0923-2508(02)01319-0 PubMed DOI

Sedlar, K. , Kolek, J. , Gruber, M. , Jureckova, K. , Branska, B. , Csaba, G. , Vasylkivska, M. , Zimmer, R. , Patakova, P. , & Provaznik, I. (2019). A transcriptional response of Clostridium beijerinckii NRRL B‐598 to a butanol shock. Biotechnology for Biofuels, 12(1), 243 10.1186/s13068-019-1584-7 PubMed DOI PMC

Sedlar, K. , Kolek, J. , Provaznik, I. , & Patakova, P. (2017). Reclassification of non‐type strain Clostridium pasteurianum NRRL B‐598 as Clostridium beijerinckii NRRL B‐598. Journal of Biotechnology, 244, 1–3. 10.1016/j.jbiotec.2017.01.003 PubMed DOI

Sedlar, K. , Kolek, J. , Skutkova, H. , Branska, B. , Provaznik, I. , & Patakova, P. (2015). Complete genome sequence of Clostridium pasteurianum NRRL B‐598, a non‐type strain producing butanol. Journal of Biotechnology, 214, 113–114. 10.1016/j.jbiotec.2015.09.022 PubMed DOI

Sedlar, K. , Koscova, P. , Vasylkivska, M. , Branska, B. , Kolek, J. , Kupkova, K. , Patakova, P. , & Provaznik, I. (2018). Transcription profiling of butanol producer Clostridium beijerinckii NRRL B‐598 using RNA‐Seq. BMC Genomics, 19(1), 415 10.1186/s12864-018-4805-8 PubMed DOI PMC

Shi, Z. , & Blaschek, H. P. (2008). Transcriptional analysis of Clostridium beijerinckii NCIMB 8052 and the hyper‐butanol‐producing mutant BA101 during the shift from acidogenesis to solventogenesis. Applied and Environmental Microbiology, 74(24), 7709–7714. 10.1128/aem.01948-08 PubMed DOI PMC

Sikkema, J. , de Bont, J. A. , & Poolman, B. (1995). Mechanisms of membrane toxicity of hydrocarbons. Microbiological Reviews, 59(2), 201–222. PubMed PMC

Tomas, C. A. , Beamish, J. , & Papoutsakis, E. T. (2004). Transcriptional analysis of butanol stress and tolerance in Clostridium acetobutylicum . Journal of Bacteriology, 186(7), 2006–2018. 10.1128/JB.186.7.2006-2018.2004 PubMed DOI PMC

Tomas, C. A. , Welker, N. E. , & Papoutsakis, E. T. (2003). Overexpression of groESL in Clostridium acetobutylicum results in increased solvent production and tolerance, prolonged metabolism, and changes in the cell's transcriptional program. Applied and Environmental Microbiology, 69(8), 4951–4965. 10.1128/AEM.69.8.4951-4965.2003 PubMed DOI PMC

Vasylkivska, M. , Jureckova, K. , Branska, B. , Sedlar, K. , Kolek, J. , Provaznik, I. , & Patakova, P. (2019). Transcriptional analysis of amino acid, metal ion, vitamin and carbohydrate uptake in butanol‐producing Clostridium beijerinckii NRRL B‐598. PLoS One, 14(11), e0224560 10.1371/journal.pone.0224560 PubMed DOI PMC

Vollherbst Schneck, K. , Sands, J. A. , & Montenecourt, B. S. (1984). Effect of butanol on lipid composition and fluidity of Clostridium acetobutylicum ATCC 824. Applied and Environmental Microbiology, 47, 193–194. PubMed PMC

Zhao, Y. , Hindorff, L. A. , Chuang, A. , Monroe‐Augustus, M. , Lyristis, M. , Harrison, M. L. , Rudolph, F. B. , & Bennett, G. N. (2003). Expression of a cloned cyclopropane fatty acid synthase gene reduces solvent formation in Clostridium acetobutylicum ATCC 824. Applied and Environmental Microbiology, 69(5), 2831–2841. 10.1128/AEM.69.5.2831-2841.2003 PubMed DOI PMC

Zuber, U. , Drzewiecki, K. , & Hecker, M. (2001). Putative sigma factor SigI (YkoZ) of Bacillus subtilis is induced by heat shock. Journal of Bacteriology, 183(4), 1472–1475. 10.1128/JB.183.4.1472-1475.2001 PubMed DOI PMC

Identification and Validation of Reference Genes in Clostridium beijerinckii NRRL B-598 for RT-qPCR Using RNA-Seq Data