Abstract
The gram-positive bacterium Clostridium thermocellum contains a set of carbohydrate-active enzymes that can potentially be employed to generate high-value-added products from lignocellulose. In this study, the gene expression profiling of C. thermocellum B8 was provided during growth in the presence of sugarcane bagasse and straw as a carbon source in comparison to growth using microcrystalline cellulose. A total of 625 and 509 genes were up-regulated for growth in the presence of bagasse and straw, respectively. These genes were mainly grouped into carbohydrate-active enzymes (CAZymes), cell motility, chemotaxis, quorum sensing pathway and expression control of glycoside hydrolases. These results show that type of carbon source modulates the gene expression profiling of carbohydrate-active enzymes. In addition, highlight the importance of cell motility, attachment to the substrate and communication in deconstructing complex substrates. This present work may contribute to the development of enzymatic cocktails and industrial strains for biorefineries based on sugarcane residues as feedstock.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
Sequences were deposited in the Sequence Read Archive (SRA) of the National Center for Biotechnology under BioProject number PRJNA625501. Raw sequence reads can be found in https://www.ncbi.nlm.nih.gov/sra/PRJNA625501.
References
Ait N, Creuzet N, Cattaneo J (1982) Properties of -Glucosidase Purified from Clostridium thermocellum. Microbiology 128:569–577. https://doi.org/10.1099/00221287-128-3-569
Akinosho H, Yee K, Close D, Ragauskas A (2014) The emergence of Clostridium thermocellum as a high utility candidate for consolidated bioprocessing applications. Front Chem 2:1–18. https://doi.org/10.3389/fchem.2014.00066
Alahuhta M, Luo Y, Ding SY, Himmel ME, Lunin VV (2011) Structure of CBM4 from Clostridium thermocellum cellulase K. Acta Crystallogr. Sect F Struct Biol Cryst Commun 67:527–530. https://doi.org/10.1107/S1744309111003307
Anbar M, Gul O, Lamed R, Sezerman UO, Bayer EA (2012) Improved thermostability of Clostridium thermocellum endoglucanase Cel8A by using consensus-guided mutagenesis. Appl Environ Microbiol 78:3458–3464. https://doi.org/10.1128/AEM.07985-11/ASSET/2DB60B26-B89C-4C75-B624-A805812BCE44/ASSETS/GRAPHIC/ZAM9991032380008.JPEG
Bahari L, Gilad Y, Borovok I, Kahel-Raifer H, Dassa B, Nataf Y, Shoham Y, Lamed R, Bayer EA (2011) Glycoside hydrolases as components of putative carbohydrate biosensor proteins in Clostridium thermocellum. J Ind Microbiol Biotechnol 38:825–832. https://doi.org/10.1007/s10295-010-0848-9
Bayer EA, Morag E, Lamed R (1994) The cellulosome — A treasure-trove for biotechnology. Trends Biotechnol 12:379–386. https://doi.org/10.1016/0167-7799(94)90039-6
Bayer EA, Shimon LJ, Shoham Y, Lamed R (1998) Cellulosomes-structure and ultrastructure. J Struct Biol 124:221–234. https://doi.org/10.1006/jsbi.1998.4065
Berger E, Zhang D, Zverlov VV, Schwarz WH (2007) Two noncellulosomal cellulases of Clostridium thermocellum, Cel9I and Cel48Y, hydrolyse crystalline cellulose synergistically. FEMS Microbiol Lett 268:194–201. https://doi.org/10.1111/j.1574-6968.2006.00583.x
Beukes N, Pletschke BI (2011) Effect of alkaline pre-treatment on enzyme synergy for efficient hemicellulose hydrolysis in sugarcane bagasse. Bioresour Technol 102:5207–5213. https://doi.org/10.1016/j.biortech.2011.01.090
Blum DL, Kataeva IA, Li X-L, Ljungdahl LG (2000) Feruloyl esterase activity of the clostridium thermocellum cellulosome can be attributed to previously unknown domains of XynY and XynZ. J Bacteriol 182(5):1346–1351
Chakraborty S, Fernandes VO, Dias FMV, Prates JAM, Ferreira LMA, Fontes CMGA, Goyal A, Centeno MSJ (2015) Role of pectinolytic enzymes identified in Clostridium thermocellum cellulosome. PLoS ONE 10:1–18. https://doi.org/10.1371/journal.pone.0116787
Cherubini F (2010) The biorefinery concept: Using biomass instead of oil for producing energy and chemicals. Energy Convers Manag 51:1412–1421. https://doi.org/10.1016/j.enconman.2010.01.015
Chundawat SPS, Beckham GT, Himmel ME, Dale BE (2011) Deconstruction of lignocellulosic biomass to fuels and chemicals. Annu Rev Chem Biomol Eng 2:121–145. https://doi.org/10.1146/annurev-chembioeng-061010-114205
de Camargo BR, Claassens NJ, Quirino BF, Noronha EF, Kengen SWM (2018) Heterologous expression and characterization of a putative glycoside hydrolase family 43 arabinofuranosidase from Clostridium thermocellum B8. Enzyme Microb Technol 109:74–83. https://doi.org/10.1016/j.enzmictec.2017.09.014
Dredge R, Radloff SE, Dyk JS, Pletschke BI (2011) Lime pretreatment of sugar beet pulp and evaluation of synergy between ArfA, ManA and XynA from Clostridium cellulovorans on the pretreated substrate. 3 Biotech 1:151–159. https://doi.org/10.1007/s13205-011-0019-3
Dror TW, Morag E, Rolider A, Bayer EA, Lamed R, Shoham Y (2003) Regulation of the Cellulosomal celS ( cel48A ) Gene of Clostridium thermocellum Is Growth Rate Dependent Regulation of the Cellulosomal celS ( cel48A ) Gene of Clostridium thermocellum Is Growth Rate Dependent. J Bacteriol 185:3042–3048. https://doi.org/10.1128/JB.185.10.3042
Dumitrache A, Klingeman DM, Natzke J, Rodriguez M Jr, Giannone RJ, Hettich RL, Davison BH, Brown SD (2017) Specialized activities and expression differences for Clostridium thermocellum biofilm and planktonic cells. Sci Rep 7:43583. https://doi.org/10.1038/srep43583
Freier D, Mothershed CP, Wiegel J (1988) Characterization of Clostridium thermocellum JW20. Appl Environ Microbiol 54:204–211
Gold ND, Martin VJJ (2007) Global view of the Clostridium thermocellum cellulosome revealed by quantitative proteomic analysis. J Bacteriol 189:6787–6795. https://doi.org/10.1128/JB.00882-07
Gonçalves GAL, Takasugi Y, Jia L, Mori Y, Noda S, Tanaka T, Ichinose H, Kamiya N (2015) Synergistic effect and application of xylanases as accessory enzymes to enhance the hydrolysis of pretreated bagasse. Enzyme Microb Technol 72:16–24. https://doi.org/10.1016/j.enzmictec.2015.01.007
Grepinet O, Chebrou M-C, Beguin P (1988) Nucleotide Sequence and Deletion Analysis of the Xylanase Gene (xynZ) of Clostridium thermocellum. J Bacteriol 170(10):4582–4588
Gulbrandsen TA, Johnsen IA, Opedal MT, Toven K, Oyaas K, Pranovich A, Mikkola JP, Hoff BH (2015) Extracting hemicelluloses from softwood and bagasse as oligosaccharides using pure water and microwave heating. Cellul Chem Technol 49:117–126
Hamann PRV, Serpa DL, Barreto da Cunha AS, de Camargo BR, Osiro KO, Valle de Sousa M, Felix CR, Miller RNG, Noronha EF (2015) Evaluation of plant cell wall degrading enzyme production by Clostridium thermocellum B8 in the presence of raw agricultural wastes. Int Biodeterior Biodegradation 105:97–105. https://doi.org/10.1016/j.ibiod.2015.08.013
Hayashi H, Takehara M, Hattori T, Kimura T, Karita S, Sakka K, Ohmiya K (1999) Nucleotide sequences of two contiguous and highly homologous xylanase genes xynA and xynB and characterization of XynA from Clostridium thermocellum. Appl Microbiol Biotechnol 51:348–357. https://doi.org/10.1007/s002530051401
Heinze S, Mechelke M, Kornberger P, Liebl W, Schwarz WH, Zverlov VV (2017) Identification of endoxylanase XynE from Clostridium thermocellum as the first xylanase of glycoside hydrolase family GH141 OPEN. Sci Rep. https://doi.org/10.1038/s41598-017-11598-y
Huang XF, Nazarian F, Vincken JP, Visser RGF, Trindade LM (2017) A tandem CBM25 domain of α-amylase from Microbacterium aurum as potential tool for targeting proteins to starch granules during starch biosynthesis. BMC Biotechnol 17:1–8. https://doi.org/10.1186/s12896-017-0406-x
Kahel-Raifer H, Jindou S, Bahari L, Nataf Y, Shoham Y, Bayer EA, Borovok I, Lamed R (2010) The unique set of putative membrane-associated anti-?? factors in Clostridium thermocellum suggests a novel extracellular carbohydrate-sensing mechanism involved in gene regulation. FEMS Microbiol Lett 308:84–93. https://doi.org/10.1111/j.1574-6968.2010.01997.x
Kataeva I, Li X, Chen H, Choi S, Ljungdahl LG (1999) Cloning and sequence analysis of a new cellulase gene encoding celk, a major cellulosome component of clostridium thermocellum : evidence for gene duplication and recombination. J Bacteriol 181(17):5288–5295
Katayeva IA, Golovchenko NP, Chuvilskaya NA, Akimenko VK (1992) Clostridium thermocellum β-glucosidases A and B: Purification, properties, localization, and regulation of biosynthesis. Enzyme Microb Technol 14:407–412. https://doi.org/10.1016/0141-0229(92)90011-C
Kopylova E, Noe L, Touzet H (2012) SortMeRNA: Fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics 28:3211–3217. https://doi.org/10.1093/bioinformatics/bts611
Kruus K, Wang WK, Ching J, Wu JHD, Kataeva IA, Seidel RD, Li XL, Ljungdahl LG, Kruus K, Wang WK, Ching J, Wu JHD (1995) Exoglucanase activities of the recombinant Clostridium thermocellum CelS, a major cellulosome component. J Bacteriol 177:1641–1644
Leis B, Held C, Bergkemper F, Dennemarck K, Steinbauer R, Reiter A, Mechelke M, Moerch M, Graubner S, Liebl W, Schwarz WH, Zverlov VV (2017) Comparative characterization of all cellulosomal cellulases from Clostridium thermocellum reveals high diversity in endoglucanase product formation essential for complex activity. Biotechnol Biofuels 10:1–16. https://doi.org/10.1186/s13068-017-0928-4
Leitão VO, Noronha EF, Camargo BR, Hamann PRV, Steindorff AS, Quirino BF, de Sousa MV, Ulhoa CJ, Felix CR (2017) Growth and expression of relevant metabolic genes of Clostridium thermocellum cultured on lignocellulosic residues. J Ind Microbiol Biotechnol 44:825–834. https://doi.org/10.1007/s10295-017-1915-2
Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. https://doi.org/10.1186/s13059-014-0550-8
Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS (2002) Microbial Cellulose Utilization: Fundamentals and Biotechnology. Microbiol Mol Biol R 66:506–577. https://doi.org/10.1128/MMBR.66.3.506-577.2002
Lyon GJ, Novick RP (2004) Peptide signaling in Staphylococcus aureus and other Gram-positive bacteria. Peptides 25:1389–1403. https://doi.org/10.1016/j.peptides.2003.11.026
Maier B, Potter L, So M, Seifert HS, Sheetz MP (2002) Single pilus motor forces exceed 100 pN. Proc Natl Acad Sci U S A 99:16012–16017. https://doi.org/10.1073/pnas.242523299
Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. Embnet. J 17:10–12. https://doi.org/10.14806/ej.17.1.200
McClure R, Balasubramanian D, Sun Y, Bobrovskyy M, Sumby P, Genco CA, Vanderpool CK, Tjaden B (2013) Computational analysis of bacterial RNA-Seq data. Nucleic Acids Res 41:1–16. https://doi.org/10.1093/nar/gkt444
Muñoz-Gutiérrez I, De Ora LO, Grinberg IR, Garty Y, Bayer EA, Shoham Y, Lamed R, Borovok I, Mu I, Ora LOD, Grinberg IR, Garty Y (2016) Decoding biomass-sensing regulons of clostridium thermocellum alternative sigma-I factors in a heterologous bacillus subtilis host system. PLoS ONE. https://doi.org/10.1371/journal.pone.0146316
Nataf Y, Bahari L, Kahel-raifer H, Borovok I, Lamed R, Bayer EA (2010) Clostridium thermocellum cellulosomal genes are regulated by extracytoplasmic polysaccharides via alternative sigma factors. Proc Nat Acad Sci. https://doi.org/10.1073/pnas.1012175107
Ng TK, Weimer PJ, Zeikus JG (1977) Cellulolytic and physiological properties of Clostridium thermocellum. Arch Microbiol 114:1–7. https://doi.org/10.1007/BF00429622
Osiro KO, de Camargo BR, Satomi R, Hamann PRV, Silva JP, de Valle SM, Quirino BF, Aquino EN, Felix CR, Murad AM, Noronha EF (2017) Characterization of Clostridium thermocellum (B8) secretome and purified cellulosomes for lignocellulosic biomass degradation. Enzyme Microb Technol 97:43–54. https://doi.org/10.1016/j.enzmictec.2016.11.002
Petre D, Millet J, Longin R, Beguin P, Girard H, Aubert JP (1986) Purification and properties of the endoglucanase C of Clostridium thermocellum produced in Escherichia coli. Biochimie 68:687–695
Pitman S, Cho KH (2015) The mechanisms of virulence regulation by small noncoding RNAs in low GC gram-positive pathogens. Int J Mol Sci 16:29797–29814. https://doi.org/10.3390/ijms161226194
Raman B, McKeown CK, Rodriguez M, Brown SD, Mielenz JR (2011) Transcriptomic analysis of Clostridium thermocellum ATCC 27405 cellulose fermentation. BMC Microbiol 11:134. https://doi.org/10.1186/1471-2180-11-134
Sand A, Holwerda EK, Ruppertsberger NM, Maloney M, Olson DG, Nataf Y, Borovok I, Sonenshein AL, Bayer EA, Lamed R, Lynd LR, Shoham Y (2015) Three cellulosomal xylanase genes in Clostridium thermocellum are regulated by both vegetative SigA (σA) and alternative SigI6 (σI6) factors. FEBS Lett 589:3133–3140. https://doi.org/10.1016/j.febslet.2015.08.026
Schmieder R, Edwards R (2011) Quality control and preprocessing of metagenomic datasets. Bioinformatics 27:863–864. https://doi.org/10.1093/bioinformatics/btr026
Schwarz WH (2001) The cellulosome and cellulose degradation by anaerobic bacteria. Appl Microbiol Biotechnol 56:634–649. https://doi.org/10.1007/s002530100710
Shoham Y, Gilad R, Gilbert HJ, Yaron S, Bayer EA, Rabinovich L, Lamed R (2003) CelI, a noncellulosomal family 9 enzyme from clostridium thermocellum, is a processive endoglucanase that degrades crystalline cellulose. J Bacteriol 185:391–398. https://doi.org/10.1128/jb.185.2.391-398.2003
Silva LF, Taciro MK, Raicher G, Piccoli RAM, Mendonça TT, Lopes MSG, Gomez JGC (2014) Perspectives on the production of polyhydroxyalkanoates in biorefineries associated with the production of sugar and ethanol. Int J Biol Macromol 71:2–7. https://doi.org/10.1016/J.IJBIOMAC.2014.06.065
Verma AK, Goyal A (2016) A novel member of family 30 glycoside hydrolase subfamily 8 glucuronoxylan endo-β-1,4-xylanase (CtXynGH30) from Clostridium thermocellum orchestrates catalysis on arabinose decorated xylans. J Mol Catal B Enzym 129:6–14. https://doi.org/10.1016/J.MOLCATB.2016.04.001
Wei H, Fu Y, Magnusson L, Baker JO, Maness PC, Xu Q, Yang S, Bowersox A, Bogorad I, Wang W, Tucker MP, Himmel ME, Ding SY (2014) Comparison of transcriptional profiles of Clostridium thermocellum grown on cellobiose and pretreated yellow poplar using RNA-seq. Front Microbiol 5:1–16. https://doi.org/10.3389/fmicb.2014.00142
Wilson CM, Rodriguez M, Johnson CM, Martin SL, Chu TM, Wolfinger RD, Hauser LJ, Land ML, Klingeman DM, Syed MH, Ragauskas AJ, Tschaplinski TJ, Mielenz JR, Brown SD (2013) Global transcriptome analysis of Clostridium thermocellum ATCC 27405 during growth on dilute acid pretreated Populus and switchgrass. Biotechnol Biofuels 6:179. https://doi.org/10.1186/1754-6834-6-179
Xu Q, Resch MG, Podkaminer K, Yang S, Baker JO, Donohoe BS, Wilson C, Klingeman DM, Olson DG, Decker SR, Giannone RJ, Hettich RL, Brown SD, Lynd LR, Bayer EA, Himmel ME, Bomble YJ (2016) Cell Biology: Dramatic performance of Clostridium thermocellum explained by its wide range of cellulase modalities. Sci Adv. https://doi.org/10.1126/sciadv.1501254
Acknowledgements
Not applicable
Funding
This study was supported by grants from the CAPES, CNPq, and FAPDF. CNPq provided scholarship for BR and EFN (Grant No. 309338/2017–4).
Author information
Authors and Affiliations
Contributions
BR and EFN, conceived the project and designed the experiments. All the authors contributed to carry the experiments, write the manuscript and agreed to publish it.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.
Ethical approval
Not applicable.
Consent for publication
Not apllicable.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
de Camargo, B.R., Steindorff, A.S., da Silva, L.A. et al. Expression profiling of Clostridium thermocellum B8 during the deconstruction of sugarcane bagasse and straw. World J Microbiol Biotechnol 39, 105 (2023). https://doi.org/10.1007/s11274-023-03546-y
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11274-023-03546-y