Transcriptional analysis of amino acid, metal ion, vitamin and carbohydrate uptake in butanol-producing Clostridium beijerinckii NRRL B-598

Autoři: Maryna Vasylkivska aff001;  Katerina Jureckova aff002;  Barbora Branska aff001;  Karel Sedlar aff002;  Jan Kolek aff001;  Ivo Provaznik aff002;  Petra Patakova aff001
Působiště autorů: Department of Biotechnology, University of Chemistry and Technology Prague, Prague, Czech Republic aff001;  Department of Biomedical Engineering, Brno University of Technology, Brno, Czech Republic aff002
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224560


In-depth knowledge of cell metabolism and nutrient uptake mechanisms can lead to the development of a tool for improving acetone-butanol-ethanol (ABE) fermentation performance and help to overcome bottlenecks in the process, such as the high cost of substrates and low production rates. Over 300 genes potentially encoding transport of amino acids, metal ions, vitamins and carbohydrates were identified in the genome of the butanol-producing strain Clostridium beijerinckii NRRL B-598, based on similarity searches in protein function databases. Transcriptomic data of the genes were obtained during ABE fermentation by RNA-Seq experiments and covered acidogenesis, solventogenesis and sporulation. The physiological roles of the selected 81 actively expressed transport genes were established on the basis of their expression profiles at particular stages of ABE fermentation. This article describes how genes encoding the uptake of glucose, iron, riboflavin, glutamine, methionine and other nutrients take part in growth, production and stress responses of C. beijerinckii NRRL B-598. These data increase our knowledge of transport mechanisms in solventogenic Clostridium and may be used in the selection of individual genes for further research.

Klíčová slova:

Carbohydrates – Clostridium – Fermentation – Glucose – Vitamins – Zinc – Butanol – Riboflavin


1. Xing W, Xu G, Dong J, Han R, Ni Y. Novel dihydrogen-bonding deep eutectic solvents: Pretreatment of rice straw for butanol fermentation featuring enzyme recycling and high solvent yield. Chem Eng J. 2018;333: 712–720. doi: 10.1016/j.cej.2017.09.176

2. Algayyim SJM, Wandel AP, Yusaf T, Hamawand I. Production and application of ABE as a biofuel. Renew Sustain Energy Rev. 2018;82: 1195–1214. doi: 10.1016/j.rser.2017.09.082

3. Luo H, Zhang J, Wang H, Chen R, Shi Z, Li X, et al. Effectively enhancing acetone concentration and acetone/butanol ratio in ABE fermentation by a glucose/acetate co-substrate system incorporating with glucose limitation and C. acetobutylicum/S. cerevisiae co-culturing. Biochem Eng J. 2017;118: 132–142. doi: 10.1016/j.bej.2016.12.003

4. Mascal M. Chemicals from biobutanol: Technologies and markets. Biofuel Bioprod Biorefin. 2012;6: 483–493. doi: 10.1002/bbb.1328

5. Prat D, Pardigon O, Flemming HW, Letestu S, Ducandas V, Isnard P, et al. Sanofi’s solvent selection guide: A step toward more sustainable processes. Org Process Res Dev. 2013;17: 1517–1525. doi: 10.1021/op4002565

6. Byrne FP, Jin S, Paggiola G, Petchey THM, Clark JH, Farmer TJ, et al. Tools and techniques for solvent selection: green solvent selection guides. Sustain Chem Process. 2016;4: 7. doi: 10.1186/s40508-016-0051-z

7. Luo H, Ge L, Zhang J, Zhao Y, Ding J, Li Z, et al. Enhancing butanol production under the stress environments of co-culturing Clostridium acetobutylicum/Saccharomyces cerevisiae integrated with exogenous butyrate addition. PLoS One. 2015;10: e0141160. doi: 10.1371/journal.pone.0141160 26489085

8. Wu YD, Xue C, Chen LJ, Bai FW. Effect of zinc supplementation on acetone-butanol-ethanol fermentation by Clostridium acetobutylicum. J Biotechnol. 2013;165: 18–21. doi: 10.1016/j.jbiotec.2013.02.009 23458964

9. Liao Z, Suo Y, Xue C, Fu H, Wang J. Improving the fermentation performance of Clostridium acetobutylicum ATCC 824 by strengthening the VB1 biosynthesis pathway. Appl Microbiol Biotechnol. 2018;102: 8107–8119. doi: 10.1007/s00253-018-9208-x 29987383

10. Yang Y, Lang N, Yang G, Yang S, Jiang W, Gu Y. Improving the performance of solventogenic clostridia by reinforcing the biotin synthetic pathway. Metab Eng. 2016;35: 121–128. doi: 10.1016/j.ymben.2016.02.006 26924180

11. Patakova P, Lipovsky J, Cizkova H, Fortova J, Rychtera M, Melzoch K. Exploitation of food feedstock and waste for production of biobutanol. Czech J Food Sci. 2009;27: 276–283. doi: 10.2144/000113087

12. Mitchell WJ. Sugar uptake by the solventogenic clostridia. World J Microbiol Biotechnol. 2016;32: 32. doi: 10.1007/s11274-015-1981-4 26748809

13. Servinsky MD, Kiel JT, Dupuy NF, Sund CJ. Transcriptional analysis of differential carbohydrate utilization by Clostridium acetobutylicum. Microbiology. 2010;156: 3478–91. doi: 10.1099/mic.0.037085-0 20656779

14. Al Makishah NH, Mitchell WJ. Dual substrate specificity of an n-acetylglucosamine phosphotransferase system in Clostridium beijerinckii. Appl Environ Microbiol. 2013;79: 6712–6718. doi: 10.1128/AEM.01866-13 23995920

15. Reeve BWP, Reid SJ. Glutamate and histidine improve both solvent yields and the acid tolerance response of Clostridium beijerinckii NCP 260. J Appl Microbiol. 2016;120: 1271–1281. doi: 10.1111/jam.13067 26789025

16. Sedlar K, Kolek J, Provaznik I, Patakova P. Reclassification of non-type strain Clostridium pasteurianum NRRL B-598 as Clostridium beijerinckii NRRL B-598. J Biotechnol. 2017;244: 1–3. doi: 10.1016/j.jbiotec.2017.01.003 28111164

17. Sedlar K, Kolek J, Skutkova H, Branska B, Provaznik I, Patakova P. Complete genome sequence of Clostridium pasteurianum NRRL B-598, a non-type strain producing butanol. J Biotechnol. 2015;214: 113–114. doi: 10.1016/j.jbiotec.2015.09.022 26410453

18. Kolek J, Sedlar K, Provaznik I, Patakova P. Dam and Dcm methylations prevent gene transfer into Clostridium pasteurianum NRRL B-598: development of methods for electrotransformation, conjugation, and sonoporation. Biotechnol Biofuels. 2016;9: 14. doi: 10.1186/s13068-016-0436-y 26793273

19. Kolek J, Branska B, Drahokoupil M, Patakova P, Melzoch K. Evaluation of viability, metabolic activity and spore quantity in clostridial cultures during ABE fermentation. FEMS Microbiol Lett. 2016;363: fnw031. doi: 10.1093/femsle/fnw031 26862145

20. Patakova P, Branska B, Sedlar K, Vasylkivska M, Jureckova K, Kolek J, et al. Acidogenesis, solventogenesis, metabolic stress response and life cycle changes in Clostridium beijerinckii NRRL B-598 at the transcriptomic level. Sci Rep. 2019;9: 1371. doi: 10.1038/s41598-018-37679-0 30718562

21. Sedlar K, Koscova P, Vasylkivska M, Branska B, Kolek J, Kupkova K, et al. Transcription profiling of butanol producer Clostridium beijerinckii NRRL B-598 using RNA-Seq. BMC Genom. 2018;19: 415. doi: 10.1186/s12864-018-4805-8 29843608

22. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30: 2114–2120. doi: 10.1093/bioinformatics/btu170 24695404

23. Kopylova E, Noe L, Touzet H. SortMeRNA: Fast and accurate filtering of ribosomal RNAs in metatranscriptomic data. Bioinformatics. 2012;28: 3211–3217. doi: 10.1093/bioinformatics/bts611 23071270

24. Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Res. 2013;41: 590–596. doi: 10.1093/nar/gks1219 23193283

25. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: Ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29: 15–21. doi: 10.1093/bioinformatics/bts635 23104886

26. Liao Y, Smyth GK, Shi W. FeatureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30: 923–930. doi: 10.1093/bioinformatics/btt656 24227677

27. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15: 550. doi: 10.1186/s13059-014-0550-8 25516281

28. Robinson MD, McCarthy DJ, Smyth GK. EdgeR: A Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26: 139–140. doi: 10.1093/bioinformatics/btp616 19910308

29. Bateman A, Martin MJ, O’Donovan C, Magrane M, Apweiler R, Alpi E, et al. UniProt: A hub for protein information. Nucleic Acids Res. 2015;43: D204–D212. doi: 10.1093/nar/gku989 25348405

30. Mitchell AL, Attwood TK, Babbitt PC, Blum M, Bork P, Bridge A, et al. InterPro in 2019: Improving coverage, classification and access to protein sequence annotations. Nucleic Acids Res. 2019;47: D351–D360. doi: 10.1093/nar/gky1100 30398656

31. El-Gebali S, Mistry J, Bateman A, Eddy SR, Luciani A, Potter SC, et al. The Pfam protein families database in 2019. Nucleic Acids Res. 2019;47: D427–D432. doi: 10.1093/nar/gky995 30357350

32. Gish W, States DJ. Identification of protein coding regions by database similarity search. Nat Genet. 1993;3: 266–272. doi: 10.1038/ng0393-266 8485583

33. Saxena J, Tanner RS. Optimization of a corn steep medium for production of ethanol from synthesis gas fermentation by Clostridium ragsdalei. World J Microbiol Biotechnol. 2012;28: 1553–1561. doi: 10.1007/s11274-011-0959-0 22805937

34. Soni BK, Soucaille P, Goma G. Continuous acetone-butanol fermentation: a global approach for the improvement in the solvent productivity in synthetic medium. Appl Microbiol Biotechnol. 1987;25: 317–321. doi: 10.1007/bf00252540

35. Nguyen NPT, Raynaud C, Meynial-Salles I, Soucaille P. Reviving the Weizmann process for commercial n-butanol production. Nat Commun. 2018;9: 3682. doi: 10.1038/s41467-018-05661-z 30206218

36. Long S, Jones DT, Woods DR. Sporulation of Clostridium acetobutylicum P262 in a defined medium. Appl Environ Microbiol. 1983;45: 1389–1393. 16346276

37. Storari M, Kulli S, Wüthrich D, Bruggmann R, Berthoud H, Arias-Roth E. Genomic approach to studying nutritional requirements of Clostridium tyrobutyricum and other Clostridia causing late blowing defects. Food Microbiol. 2016;59: 213–223. doi: 10.1016/ 27375262

38. Jiao S, Zhang Y, Wan C, Lv J, Du R, Zhang R, et al. Transcriptional analysis of degenerate strain Clostridium beijerinckii DG-8052 reveals a pleiotropic response to CaCO(3)-associated recovery of solvent production. Sci Rep. 2016;6: 38818. doi: 10.1038/srep38818 27966599

39. Alsaker K V., Paredes C, Papoutsakis ET. 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. Biotechnol Bioeng. 2010;105: 1131–1147. doi: 10.1002/bit.22628 19998280

40. Amador-Noguez D, Brasg IA, Feng X-J, Roquet N, Rabinowitz JD. Metabolome remodeling during the acidogenic-solventogenic transition in Clostridium acetobutylicum. Appl Environ Microbiol. 2011;77: 7984–7997. doi: 10.1128/AEM.05374-11 21948824

41. Alsaker K V, Papoutsakis ET. Transcriptional program of early sporulation and stationary-phase events in Clostridium acetobutylicum. J Bacteriol. 2005;187: 7103–7118. doi: 10.1128/JB.187.20.7103-7118.2005 16199581

42. Poudel S, Giannone RJ, Rodriguez M, Raman B, Martin MZ, Engle NL, et al. Integrated omics analyses reveal the details of metabolic adaptation of Clostridium thermocellum to lignocellulose-derived growth inhibitors released during the deconstruction of switchgrass. Biotechnol Biofuels. 2017;10: 14. doi: 10.1186/s13068-016-0697-5 28077967

43. Henson MA, Phalak P. In silico analysis of Clostridium difficile biofilm metabolism and treatment. IFAC-PapersOnLine. 2016;49: 153–158. doi: 10.1016/j.ifacol.2016.12.118

44. Larocque M, Chenard T, Najmanovich R. A curated C. difficile strain 630 metabolic network: prediction of essential targets and inhibitors. BMC Syst Biol. 2014;8: 117. doi: 10.1186/s12918-014-0117-z 25315994

45. Heluane H, Dagher MRE, Bruno-Barcena JM. Meta-analysis and functional validation of nutritional requirements of solventogenic clostridia growing under butanol stress conditions and coutilization of D-glucose and D-xylose. Appl Environ Microbiol. 2011;77: 4473–4485. doi: 10.1128/AEM.00116-11 21602379

46. Luo H, Ge L, Zhang J, Ding J, Chen R, Shi Z. Enhancing acetone biosynthesis and acetone–butanol–ethanol fermentation performance by co-culturing Clostridium acetobutylicum/Saccharomyces cerevisiae integrated with exogenous acetate addition. Bioresour Technol. 2016;200: 111–120. doi: 10.1016/j.biortech.2015.09.116 26476171

47. Neumann-Schaal M, Hofmann JD, Will SE, Schomburg D. Time-resolved amino acid uptake of Clostridium difficile 630Δerm and concomitant fermentation product and toxin formation. BMC Microbiol. 2015;15: 281. doi: 10.1186/s12866-015-0614-2 26680234

48. Durre P. Physiologyand sporulation in Clostridium. Microbiol Spectr. 2014;2: 315–329. doi: 10.1128/microbiolspec.tbs-0010-2012 26104199

49. Eisenstadt E. Potassium content during growth and sporulation in Bacillus subtilis. J Bacteriol. 1972;112: 264–267. 4627924

50. Wu YD, Xue C, Chen LJ, Wan HH, Bai FW. Transcriptional analysis of micronutrient zinc-associated response for enhanced carbohydrate utilization and earlier solventogenesis in Clostridium acetobutylicum. Sci Rep. 2015;5: 16598. doi: 10.1038/srep16598 26586044

51. Winzer K, Lorenz K, Durre P. Acetate kinase from Clostridium acetobutylicum: A highly specific enzyme that is actively transcribed during acidogenesis and solventogenesis. Microbiology. 1997;143: 3279–3286. doi: 10.1099/00221287-143-10-3279 9353928

52. Zhao X, Xing D, Liu B, Lu L, Zhao J, Ren N. The effects of metal ions and L-cysteine on hydA gene expression and hydrogen production by Clostridium beijerinckii RZF-1108. Int J Hydrogen Energy. 2012;37: 13711–13717. doi: 10.1016/j.ijhydene.2012.02.144

53. Chin HL, Chen ZS, Chou CP. Fedbatch operation using Clostridium acetobutylicum suspension culture as biocatalyst for enhancing hydrogen production. Biotechnol Prog. 2003;19: 383–388. doi: 10.1021/bp0200604 12675576

54. Bao MD, Su HJ, Tan TW. Dark fermentative bio-hydrogen production: Effects of substrate pre-treatment and addition of metal ions or L-cysteine. Fuel. 2013;112: 38–44. doi: 10.1016/j.fuel.2013.04.063

55. Wu H, Wang C, Chen P, He A-Y, Xing F-X, Kong X-P, et al. Effects of pH and ferrous iron on the coproduction of butanol and hydrogen by Clostridium beijerinckii IB4. Int J Hydrogen Energy. 2017;42: 6547–6555. doi: 10.1016/j.ijhydene.2017.02.094

56. Serio AW, Pechter KB, Sonenshein AL. Bacillus subtilis aconitase is required for efficient late-sporulation gene expression. J Bacteriol. 2006;188: 6396–6405. doi: 10.1128/JB.00249-06 16923907

57. Wu YD, Xue C, Chen LJ, Yuan WJ, Bai FW. Improvements of metabolites tolerance in Clostridium acetobutylicum by micronutrient zinc supplementation. Biotechnol Bioprocess Eng. 2016;21: 60–67. doi: 10.1007/s12257-015-0583-1

58. Zhang Y, Rodionov DA, Gelfand MS, Gladyshev VN. Comparative genomic analyses of nickel, cobalt and vitamin B12 utilization. BMC Genom. 2009;10: 78. doi: 10.1186/1471-2164-10-78 19208259

59. Paredes CJ, Rigoutsos I, Papoutsakis T. Transcriptional organization of the Clostridium acetobutylicum genome. Nucleic Acids Res. 2004;32: 1973–1981. doi: 10.1093/nar/gkh509 15060177

60. Jaehme M, Slotboom DJ. Diversity of membrane transport proteins for vitamins in bacteria and archaea. Biochim Biophys Acta—Gen Subj. 2015;1850: 565–576. doi: 10.1016/j.bbagen.2014.05.006 24836521

61. Maret W, Wedd A. Binding, transport and storage of metal ions in biological cells. Cambridge: The Royal Society of Chemistry; 2014. doi: 10.1039/9781849739979

62. Shukla G, Thakur V. Biohydrogen production from rice mill wastes by Clostridium acetobutylicum NCIM 2877. World J Eng. 2015;12: 383–390. doi: 10.1260/1708-5284.12.4.383

63. Zhang Y, Ezeji TC. Transcriptional analysis of Clostridium beijerinckii NCIMB 8052 to elucidate role of furfural stress during acetone butanol ethanol fermentation. Biotechnol Biofuels. 2013;6: 66. doi: 10.1186/1754-6834-6-66 23642190

64. Hönicke D, Janssen H, Grimmler C, Ehrenreich A, Lütke-Eversloh T. Global transcriptional changes of Clostridium acetobutylicum cultures with increased butanol: acetone ratios. N Biotechnol. 2012;29: 485–493. doi: 10.1016/j.nbt.2012.01.001 22285530

65. Worst DJ, Gerrits MM, Vandenbroucke-Grauls CMJE, Kusters JG. Helicobacter pylori ribBA-mediated riboflavin production is involved in iron acquisition. J Bacteriol. 1998;180: 1473–1479. 9515916

66. Crossley RA, Gaskin DJH, Holmes K, Mulholland F, Wells JM, Kelly DJ, et al. Riboflavin biosynthesis is associated with assimilatory ferric reduction and iron acquisition by Campylobacter jejuni. Appl Environ Microbiol. 2007;73: 7819–7825. doi: 10.1128/AEM.01919-07 17965203

67. Von Canstein H, Ogawa J, Shimizu S, Lloyd JR. Secretion of flavins by Shewanella species and their role in extracellular electron transfer. Appl Environ Microbiol. 2008;74: 615–623. doi: 10.1128/AEM.01387-07 18065612

68. Rodionov DA, Li X, Rodionova IA, Yang C, Sorci L, Dervyn E, et al. Transcriptional regulation of NAD metabolism in bacteria: Genomic reconstruction of NiaR (YrxA) regulon. Nucleic Acids Res. 2008;36: 2032–2046. doi: 10.1093/nar/gkn046 18276644

69. Li T, Yan Y, He J. Reducing cofactors contribute to the increase of butanol production by a wild-type Clostridium sp. strain BOH3. Bioresour Technol. 2014;155: 220–228. doi: 10.1016/j.biortech.2013.12.089 24463410

70. Mitchell WJ. The phosphotransferase system in solventogenic clostridia. J Mol Microbiol Biotechnol. 2015;25: 129–142. doi: 10.1159/000375125 26159074

71. Shi Y, Li Y-X, Li Y-Y. Large number of phosphotransferase genes in the Clostridium beijerinckii NCIMB 8052 genome and the study on their evolution. BMC Bioinformatics. 2010;11: S9. doi: 10.1186/1471-2105-11-S11-S9 21172059

72. Tangney M, Mitchell WJ. Characterisation of a glucose phosphotransferase system in Clostridium acetobutylicum ATCC 824. Appl Microbiol Biotechnol. 2007;74: 398–405. doi: 10.1007/s00253-006-0679-9 17096120

73. Wang Y, Li X, Blaschek HP. Effects of supplementary butyrate on butanol production and the metabolic switch in Clostridium beijerinckii NCIMB 8052: genome-wide transcriptional analysis with RNA-Seq. Biotechnol Biofuels. 2013;6: 138. doi: 10.1186/1754-6834-6-138 24229082

74. Wang Y, Li X, Mao Y, Blaschek HP. Genome-wide dynamic transcriptional profiling in Clostridium beijerinckii NCIMB 8052 using single-nucleotide resolution RNA-Seq. BMC Genom. 2012;13: 102. doi: 10.1186/1471-2164-13-102 22433311

75. Seo S-O, Janssen H, Magis A, Wang Y, Lu T, Price ND, et al. Genomic, transcriptional, and phenotypic analysis of the glucose derepressed Clostridium beijerinckii mutant exhibiting acid crash phenotype. Biotechnol J. 2017;12: 1700182. doi: 10.1002/biot.201700182 28762642

76. 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 Microbiol. 2008;74: 7709–7714. doi: 10.1128/AEM.01948-08 18849451

77. Zhang L, Leyn SA, Gu Y, Jiang W, Rodionov DA, Yang C. Ribulokinase and transcriptional regulation of arabinose metabolism in Clostridium acetobutylicum. J Bacteriol. 2012;194: 1055–1064. doi: 10.1128/JB.06241-11 22194461

78. Sun Z, Chen Y, Yang C, Yang S, Gu Y, Jiang W. A novel three-component system-based regulatory model for D-xylose sensing and transport in Clostridium beijerinckii. Mol Microbiol. 2015;95: 576–589. doi: 10.1111/mmi.12894 25441682

79. Mitchell WJ. Physiology of carbohydrate to solvent conversion by Clostridia. Adv Microb Physiol. 1997;39: 31–130. doi: 10.1016/S0065-2911(08)60015-6

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