Applied Microbiology and Biotechnology

, Volume 99, Issue 2, pp 885–896 | Cite as

Inverse metabolic engineering of Bacillus subtilis for xylose utilization based on adaptive evolution and whole-genome sequencing

  • Bo Zhang
  • Ning Li
  • Zhiwen Wang
  • Ya-Jie Tang
  • Tao ChenEmail author
  • Xueming Zhao
Applied microbial and cell physiology


Efficient utilization of xylose by bacteria is essential for production of fuels and chemicals from lignocellulosic biomass. In this study, Bacillus subtilis 168 was subjected to laboratory adaptive evolution, and a mutant E72, which could grow on xylose with a maximum specific growth rate of 0.445 h−1, was obtained. By whole-genome sequencing, 16 mutations were identified in strain E72. Through further analysis, three of them, which were in the coding regions of genes araR, sinR, and comP, were identified as the beneficial mutations. The reconstructed strain 168ARSRCP harboring these three mutations exhibited similar growth capacity on xylose to the evolved strain E72, and the average xylose consumption rate of this strain is 0.530 g/l/h, much higher than that of E72 (0.392 g/l/h). Furthermore, genes acoA and bdhA were deleted and the final strain could utilize xylose to produce acetoin at 71 % of the maximum theoretical yield. These results suggested that this strain could be used as a potential platform for production of fuels and chemicals from lignocellulosic biomass.


Bacillus subtilis Xylose Adaptive evolution Whole-genome sequencing Acetoin Inverse metabolic engineering 



This work was supported by National 973 Project [2011CBA00804, 2012CB725203]; National Natural Science Foundation of China [NSFC-21176182, NSFC-21206112, NSFC-21390201]; Natural Science Foundation of Tianjin (No. 12JCYBJC12900); the Research Fund for the Doctoral Program of Higher Education (20100032120014); the Independent Innovation of Tianjin University (No. 2010XJ-0049).

Supplementary material

253_2014_6131_MOESM1_ESM.pdf (565 kb)
ESM 1 (PDF 564 kb)


  1. Anagnostopoulos C, Spizizen J (1961) Requirements for transformation in Bacillus subtilis. J Bacteriol 81(5):741–746PubMedPubMedCentralGoogle Scholar
  2. Barbe V, Cruveiller S, Kunst F, Lenoble P, Meurice G, Sekowska A, Vallenet D, Wang T, Moszer I, Medigue C, Danchin A (2009) From a consortium sequence to a unified sequence: the Bacillus subtilis 168 reference genome a decade later. Microbiol (UK) 155:1758–1775. doi: 10.1099/mic.0.027839-0 CrossRefGoogle Scholar
  3. Butcher RA, Schroeder FC, Fischbach MA, Straightt PD, Kolter R, Walsh CT, Clardy J (2007) The identification of bacillaene, the product of the PksX megacomplex in Bacillus subtilis. Proc Natl Acad Sci U S A 104(5):1506–1509. doi: 10.1073/pnas.0610503104 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Chen T, Liu WX, Fu J, Zhang B, Tang YJ (2013) Engineering Bacillus subtilis for acetoin production from glucose and xylose mixtures. J Biotechnol 168(4):499–505. doi: 10.1016/j.jbiotec.2013.09.020 CrossRefPubMedGoogle Scholar
  5. Chillappagari S, Miethke M, Trip H, Kuipers OP, Marahiel MA (2009) Copper acquisition is mediated by YcnJ and regulated by YcnK and CsoR in Bacillus subtilis. J Bacteriol 191(7):2362–2370. doi: 10.1128/JB.01616-08 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Cox J, Schubert AM, Travisano M, Putonti C (2010) Adaptive evolution and inherent tolerance to extreme thermal environments. BMC Evol Biol 10(75):1471–2148. doi: 10.1186/1471-2148-10-75 Google Scholar
  7. Craig JE, Ford MJ, Blaydon DC, Sonenshein AL (1997) A null mutation in the Bacillus subtilis aconitase gene causes a block in Spo0A-phosphate-dependent gene expression. J Bacteriol 179(23):7351–7359CrossRefPubMedPubMedCentralGoogle Scholar
  8. Ekberg J, Rautio J, Mattinen L, Vidgren V, Londesborough J, Gibson BR (2013) Adaptive evolution of the lager brewing yeast Saccharomyces pastorianus for improved growth under hyperosmotic conditions and its influence on fermentation performance. FEMS Yeast Res 13(3):335–349. doi: 10.1111/1567-1364.12038 CrossRefPubMedGoogle Scholar
  9. Fabret C, Ehrlich SD, Noirot P (2002) A new mutation delivery system for genome-scale approaches in Bacillus subtilis. Mol Microbiol 46(1):25–36. doi: 10.1046/j.1365-2958.2002.03140.x CrossRefPubMedGoogle Scholar
  10. Fernández-Sandoval MT, Huerta-Beristain G, Trujillo-Martinez B, Bustos P, González V, Bolivar F, Gosset G, Martinez A (2012) Laboratory metabolic evolution improves acetate tolerance and growth on acetate of ethanologenic Escherichia coli under non-aerated conditions in glucose-mineral medium. Appl Microbiol Biotechnol 96(5):1291–1300. doi: 10.1007/s00253-012-4177-y CrossRefPubMedGoogle Scholar
  11. Foulger D, Errington J (1998) A 28 kbp segment from the spoVM region of the Bacillus subtilis 168 genome. Microbiology 144(Pt 3):801–805CrossRefPubMedGoogle Scholar
  12. Fu J, Wang Z, Chen T, Liu W, Shi T, Wang G, Tang Y-J, Zhao X (2014) NADH plays the vital role for chiral pure D-(−)-2,3-butanediol production in Bacillus subtilis under limited oxygen conditions. Biotechnol Bioeng. doi: 10.1002/bit.25265 Google Scholar
  13. Gaur NK, Oppenheim J, Smith I (1991) The Bacillus subtilis sin gene, a regulator of alternate developmental processes, codes for a DNA-binding protein. J Bacteriol 173(2):678–686CrossRefPubMedPubMedCentralGoogle Scholar
  14. Haima P, Bron S, Venema G (1987) The effect of restriction on shotgun cloning and plasmid stability in Bacillus subtilis Marburg. Mol Gen Genet 209(2):335–342. doi: 10.1007/BF00329663 CrossRefPubMedGoogle Scholar
  15. Hamoen LW, Venema G, Kuipers OP (2003) Controlling competence in Bacillus subtilis: shared use of regulators. Microbiology 149(Pt 1):9–17. doi: 10.1099/mic.0.26003-0 CrossRefPubMedGoogle Scholar
  16. Hanahan D (1985) Techniques for transformation of E. coli. In: Glover DM (ed) DNA cloning: a practical approach, vol 1. IRL, Oxford, pp 109–135Google Scholar
  17. Harwood CR, Cutting SM, Chambert R (1990) Molecular biological methods for Bacillus. Wiley, ChichesterGoogle Scholar
  18. Herring CD, Raghunathan A, Honisch C, Patel T, Applebee MK, Joyce AR, Albert TJ, Blattner FR, van den Boom D, Cantor CR, Palsson BO (2006) Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale. Nat Genet 38(12):1406–1412. doi: 10.1038/ng1906 CrossRefPubMedGoogle Scholar
  19. Hua Q, Joyce AR, Palsson BØ, Fong SS (2007) Metabolic characterization of Escherichia coli strains adapted to growth on lactate. Appl Environ Microbiol 73(14):4639–4647. doi: 10.1128/aem.00527-07 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Inaoka T, Matsumura Y, Tsuchido T (1999) SodA and manganese are essential for resistance to oxidative stress in growing and sporulating cells of Bacillus subtilis. J Bacteriol 181(6):1939–1943PubMedPubMedCentralGoogle Scholar
  21. Kim OC, Suwannarangsee S, Oh DB, Kim S, Seo JW, Kim CH, Kang HA, Kim JY, Kwon O (2013a) Transcriptome analysis of xylose metabolism in the thermotolerant methylotrophic yeast Hansenula polymorpha. Bioprocess Biosyst Eng 36(10):1509–1518. doi: 10.1007/s00449-013-0909-3 CrossRefPubMedGoogle Scholar
  22. Kim SR, Park Y-C, Jin Y-S, Seo J-H (2013b) Strain engineering of Saccharomyces cerevisiae for enhanced xylose metabolism. Biotechnol Adv 31(6):851–861. doi: 10.1016/j.biotechadv.2013.03.004 CrossRefPubMedGoogle Scholar
  23. Krispin O, Allmansberger R (1998) The Bacillus subtilis AraE protein displays a broad substrate specificity for several different sugars. J Bacteriol 180(12):3250–3252PubMedPubMedCentralGoogle Scholar
  24. Kuhad RC, Gupta R, Khasa YP, Singh A, Zhang YHP (2011) Bioethanol production from pentose sugars: current status and future prospects. Renew Sust Energ Rev 15(9):4950–4962. doi: 10.1016/j.rser.2011.07.058 CrossRefGoogle Scholar
  25. Lawlis VB, Dennis MS, Chen EY, Smith DH, Henner DJ (1984) Cloning and sequencing of the xylose isomerase and xylulose kinase genes of Escherichia coli. Appl Environ Microbiol 47(1):15–21PubMedPubMedCentralGoogle Scholar
  26. Lazarevic V, Soldo B, Medico N, Pooley H, Bron S, Karamata D (2005) Bacillus subtilis α-phosphoglucomutase is required for normal cell morphology and biofilm formation. Appl Environ Microbiol 71(1):39–45. doi: 10.1128/AEM.71.1.39-45.2005 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Lee D-H, Palsson BØ (2010) Adaptive evolution of Escherichia coli K-12 MG1655 during growth on a nonnative carbon source, L-1,2-propanediol. Appl Environ Microbiol 76(13):4158–4168. doi: 10.1128/aem.00373-10 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Lee J-Y, Seo J, Kim E-S, Lee H-S, Kim P (2013) Adaptive evolution of Corynebacterium glutamicum resistant to oxidative stress and its global gene expression profiling. Biotechnol Lett 35(5):709–717. doi: 10.1007/s10529-012-1135-9 CrossRefPubMedGoogle Scholar
  29. Liu Y, Liu L, Shin H-D, Chen RR, Li J, Du G, Chen J (2013) Pathway engineering of Bacillus subtilis for microbial production of N-acetylglucosamine. Metab Eng 19:107–115. doi: 10.1016/j.ymben.2013.07.002 CrossRefPubMedGoogle Scholar
  30. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method. Methods 25(4):402–408. doi: 10.1006/meth.2001.1262 CrossRefPubMedGoogle Scholar
  31. Mandic-Mulec I, Doukhan L, Smith I (1995) The Bacillus subtilis SinR protein is a repressor of the key sporulation gene spo0A. J Bacteriol 177(16):4619–4627CrossRefPubMedPubMedCentralGoogle Scholar
  32. Márquez-Magaña LM, Mirel DB, Chamberlin MJ (1994) Regulation of sigma D expression and activity by spo0, abrB, and sin gene products in Bacillus subtilis. J Bacteriol 176(8):2435–2438CrossRefPubMedPubMedCentralGoogle Scholar
  33. Meijnen J-P, de Winde JH, Ruijssenaars HJ (2008) Engineering Pseudomonas putida S12 for efficient utilization of D-xylose and L-arabinose. Appl Environ Microbiol 74(16):5031–5037. doi: 10.1128/aem.00924-08 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, Ladisch M (2005) Features of promising technologies for pretreatment of lignocellulosic biomass. Bioresour Technol 96(6):673–686. doi: 10.1016/j.biortech.2004.06.025 CrossRefPubMedGoogle Scholar
  35. Mota LJ, Tavares P, Sa-Nogueira I (1999) Mode of action of AraR, the key regulator of L-arabinose metabolism in Bacillus subtilis. Mol Microbiol 33(3):476–489. doi: 10.1046/j.1365-2958.1999.01484.x CrossRefPubMedGoogle Scholar
  36. Paik SH, Chakicherla A, Hansen JN (1998) Identification and characterization of the structural and transporter genes for, and the chemical and biological properties of, sublancin 168, a novel lantibiotic produced by Bacillus subtilis 168. J Biol Chem 273:23134–23142. doi: 10.1074/jbc.273.36.23134
  37. Park YC, Jun SY, Seo JH (2012) Construction and characterization of recombinant Bacillus subtilis JY123 able to transport xylose efficiently. J Biotechnol 161(4):402–406. doi: 10.1016/j.jbiotec.2012.07.192 CrossRefPubMedGoogle Scholar
  38. Piazza F, Tortosa P, Dubnau D (1999) Mutational analysis and membrane topology of ComP, a quorum-sensing histidine kinase of Bacillus subtilis controlling competence development. J Bacteriol 181(15):4540–4548PubMedPubMedCentralGoogle Scholar
  39. Portnoy VA, Bezdan D, Zengler K (2011) Adaptive laboratory evolution—harnessing the power of biology for metabolic engineering. Curr Opin Biotechnol 22(4):590–594. doi: 10.1016/j.copbio.2011.03.007 CrossRefPubMedGoogle Scholar
  40. Scalcinati G, Otero JM, Van Vleet JRH, Jeffries TW, Olsson L, Nielsen J (2012) Evolutionary engineering of Saccharomyces cerevisiae for efficient aerobic xylose consumption. FEMS Yeast Res 12(5):582–597. doi: 10.1111/j.1567-1364.2012.00808.x CrossRefPubMedGoogle Scholar
  41. Schiott T, von Wachenfeldt C, Hederstedt L (1997) Identification and characterization of the ccdA gene, required for cytochrome c synthesis in Bacillus subtilis. J Bacteriol 197(6):1962–1973Google Scholar
  42. Schmiedel D, Hillen W (1996) A Bacillus subtilis 168 mutant with increased xylose uptake can utilize xylose as sole carbon source. FEMS Microbiol Lett 135(2–3):175–178. doi: 10.1016/0378-1097(95)00445-9 CrossRefGoogle Scholar
  43. Schurmann M, Sprenger GA (2001) Fructose-6-phosphate aldolase is a novel class I aldolase from Escherichia coli and is related to a novel group of bacterial transaldolases. J Biol Chem 276(14):11055–11061. doi: 10.1074/jbc.M008061200 CrossRefPubMedGoogle Scholar
  44. Sekiguchi J, Ezaki B, Kodama K, Akamatsu T (1988) Molecular cloning of a gene affecting the autolysin level and flagellation in Bacillus subtilis. J Gen Microbiol 134(6):1611–1621PubMedGoogle Scholar
  45. Shen Y, Chen X, Peng B, Chen L, Hou J, Bao X (2012) An efficient xylose-fermenting recombinant Saccharomyces cerevisiae strain obtained through adaptive evolution and its global transcription profile. Appl Microbiol Biotechnol 96(4):1079–1091. doi: 10.1007/s00253-012-4418-0 CrossRefPubMedGoogle Scholar
  46. Shi S, Chen T, Zhang Z, Chen X, Zhao X (2009) Transcriptome analysis guided metabolic engineering of Bacillus subtilis for riboflavin production. Metab Eng 11(4–5):243–252. doi: 10.1016/j.ymben.2009.05.002 CrossRefPubMedGoogle Scholar
  47. Sonderegger M, Sauer U (2003) Evolutionary engineering of Saccharomyces cerevisiae for anaerobic growth on xylose. Appl Environ Microbiol 69(4):1990–1998. doi: 10.1128/aem.69.4.1990-1998.2003 CrossRefPubMedPubMedCentralGoogle Scholar
  48. Steller S, Vollenbroich D, Leenders F, Stein T, Conrad B, Hofemeister J, Jacques P, Thonart P, Vater J (1999) Structural and functional organization of the fengycin synthetase multienzyme system from Bacillus subtilis b213 and A1/3. Chem Biol 6(1):31–41. doi: 10.1016/S1074-5521(99)80018-0 CrossRefPubMedGoogle Scholar
  49. Tang MR, Sternberg D, Behr RK, Sloma A, Berka RM (2006) Use of transcriptional profiling and bioinformatics to solve production problems: eliminating red pigment production in a Bacillus subtilis strain producing hyaluronic acid. Ind Biotechnol 2(1):66–74. doi: 10.1089/ind.2006.2.66 CrossRefGoogle Scholar
  50. Utrilla J, Licona-Cassani C, Marcellin E, Gosset G, Nielsen LK, Martinez A (2012) Engineering and adaptive evolution of Escherichia coli for D-lactate fermentation reveals GatC as a xylose transporter. Metab Eng 14(5):469–476. doi: 10.1016/j.ymben.2012.07.007 CrossRefPubMedGoogle Scholar
  51. Wang Y, Manow R, Finan C, Wang J, Garza E, Zhou S (2011) Adaptive evolution of nontransgenic Escherichia coli KC01 for improved ethanol tolerance and homoethanol fermentation from xylose. J Ind Microbiol Biotechnol 38(9):1371–1377. doi: 10.1007/s10295-010-0920-5 CrossRefPubMedGoogle Scholar
  52. Wang M, Fu J, Zhang X, Chen T (2012) Metabolic engineering of Bacillus subtilis for enhanced production of acetoin. Biotechnol Lett 34(10):1877–1885. doi: 10.1007/s10529-012-0981-9 CrossRefPubMedGoogle Scholar
  53. Wilhelm M, Hollenberg CP (1985) Nucleotide sequence of the Bacillus subtilis xylose isomerase gene: extensive homology between the Bacillus and Escherichia coli enzyme. Nucleic Acids Res 13(15):5717–5722Google Scholar
  54. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33(1):103–119. doi: 10.1016/0378-1119(85)90120-9 CrossRefPubMedGoogle Scholar
  55. Zhang J, Wu C, Du G, Chen J (2012) Enhanced acid tolerance in Lactobacillus casei by adaptive evolution and compared stress response during acid stress. Biotechnol Bioprocess Eng 17(2):283–289. doi: 10.1007/s12257-011-0346-6 CrossRefGoogle Scholar
  56. Zhou H, Cheng J-S, Wang BL, Fink GR, Stephanopoulos G (2012) Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae. Metab Eng 14(6):611–622. doi: 10.1016/j.ymben.2012.07.011 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Bo Zhang
    • 1
    • 2
  • Ning Li
    • 1
    • 2
  • Zhiwen Wang
    • 1
    • 2
  • Ya-Jie Tang
    • 3
  • Tao Chen
    • 1
    • 2
    Email author
  • Xueming Zhao
    • 1
    • 2
  1. 1.Key Laboratory of Systems Bioengineering (Ministry of Education)Tianjin UniversityTianjinChina
  2. 2.SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and TechnologyTianjin UniversityTianjinChina
  3. 3.Key Laboratory of Fermentation Engineering (Ministry of Education)Hubei University of TechnologyWuhanChina

Personalised recommendations