Applied Microbiology and Biotechnology

, Volume 102, Issue 15, pp 6547–6565 | Cite as

Stabilizing displayed proteins on vegetative Bacillus subtilis cells

  • Grace L. Huang
  • Jason E. Gosschalk
  • Ye Seong Kim
  • Rachel R. Ogorzalek Loo
  • Robert T. ClubbEmail author
Applied genetics and molecular biotechnology


Microbes engineered to display heterologous proteins could be useful biotechnological tools for protein engineering, lignocellulose degradation, biocatalysis, bioremediation, and biosensing. Bacillus subtilis is a promising host to display proteins, as this model Gram-positive bacterium is genetically tractable and already used industrially to produce enzymes. To gain insight into the factors that affect displayed protein stability and copy number, we systematically compared the ability of different protease-deficient B. subtilis strains (WB800, BRB07, BRB08, and BRB14) to display a Cel8A-LysM reporter protein in which the Clostridium thermocellum Cel8A endoglucanase is fused to LysM cell wall binding modules. Whole-cell cellulase measurements and fractionation experiments demonstrate that genetically eliminating extracytoplasmic bacterial proteases improves Cel8A-LysM display levels. However, upon entering stationary phase, for all protease-deficient strains, the amount of displayed reporter dramatically decreases, presumably as a result of cellular autolysis. This problem can be partially overcome by adding chemical protease inhibitors, which significantly increase protein display levels. We conclude that strain BRB08 is well suited for stably displaying our reporter protein, as genetic removal of its extracellular and cell wall-associated proteases leads to the highest levels of surface-accumulated Cel8A-LysM without causing secretion stress or impairing growth. A two-step procedure is presented that enables the construction of enzyme-coated vegetative B. subtilis cells that retain stable cell-associated enzyme activity for nearly 3 days. The results of this work could aid the development of whole-cell display systems that have useful biotechnological applications.


Bacillus subtilis Protease-deficient Cell surface display LysM 



We thank the Bacillus Genetic Stock Center (BGSC), Cobra Biologics, and Dr. Beth Lazazzera for providing plasmids and strains, and Dr. Peter Bradley for allowing us to use his microscope. This material is based upon a work supported by the U.S. Department of Energy Office of Science, Office of Biological and Environmental Research program under Award Number DE-FC02-02ER63421. GL Huang was supported by a Ruth L. Kirschstein National Research Service Award GM007185.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9062_MOESM1_ESM.pdf (682 kb)
ESM 1 (PDF 681 kb)


  1. Anagnostopoulos C, Spizizen J (1961) Requirements for transformation in Bacillus subtilis. J Bacteriol 81(5):741–746PubMedPubMedCentralGoogle Scholar
  2. Antelmann H, Darmon E, Noone D, Veening JW, Westers H, Bron S, Kuipers OP, Devine KM, Hecker M, van Dijl JM (2003) The extracellular proteome of Bacillus subtilis under secretion stress conditions. Mol Microbiol 49(1):143–156. CrossRefPubMedGoogle Scholar
  3. Arigoni F, Talabot F, Peitsch M, Edgerton MD, Meldrum E, Allet E, Fish R, Jamotte T, Curchod ML, Loferer H (1998) A genome-based approach for the identification of essential bacterial genes. Nat Biotechnol 16(9):851–856. CrossRefPubMedGoogle Scholar
  4. Blackman SA, Smith TJ, Foster SJ (1998) The role of autolysins during vegetative growth of Bacillus subtilis 168. Microbiology 144(1):73–82. CrossRefPubMedGoogle Scholar
  5. Chen CL, Wu SC, Tjia WM, Wang CL, Lohka MJ, Wong SL (2008) Development of a LytE-based high-density surface display system in Bacillus subtilis. Microb Biotechnol 1(2):177–190. CrossRefPubMedGoogle Scholar
  6. Darmon E, Noone D, Masson A, Bron S, Kuipers OP, Devine KM, van Dijl JM (2002) A novel class of heat and secretion stress-responsive genes is controlled by the autoregulated CssRS two-component system of Bacillus subtilis. J Bacteriol 184(20):5661–5671. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Desvaux M, Dumas E, Chafsey I, Hebraud M (2006) Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure. FEMS Microbiol Lett 256(1):1–15. CrossRefPubMedGoogle Scholar
  8. Fabret C, Hoch JA (1998) A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy. J Bacteriol 180(23):6375–6383PubMedPubMedCentralGoogle Scholar
  9. Garcia-Galan C, Berenguer-Murcia A, Fernandez-Lafuente R, Rodrigues RC (2011) Potential of different enzyme immobilization strategies to improve enzyme performance. Adv Synth Catal 353(16):2885–2904. CrossRefGoogle Scholar
  10. la Grange DC, den Haan R, van Zyl WH (2010) Engineering cellulolytic ability into bioprocessing organisms. Appl Microbiol Biotechnol 87(4):1195–1208. CrossRefPubMedGoogle Scholar
  11. Harold FM (1972) Conservation and transformation of energy by bacterial membranes. Bacteriol Rev 36(2):172–230PubMedPubMedCentralGoogle Scholar
  12. Harwood CR, Cranenburgh R (2008) Bacillus protein secretion: an unfolding story. Trends Microbiol 16(2):73–79. CrossRefPubMedGoogle Scholar
  13. He XS, Shyu YT, Nathoo S, Wong SL, Doi RH (1991) Construction and use of a Bacillus subtilis mutant deficient in multiple protease genes for the expression of eukaryotic genes. Ann N Y Acad Sci 646:69–77. CrossRefPubMedGoogle Scholar
  14. Homaei AA, Sariri R, Vianello F, Stevanato R (2013) Enzyme immobilization: an update. J Chem Biol 6(4):185–205. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Huang GL, Clubb RT (2017) Progress towards engineering microbial surfaces to degrade biomass. Biomass volume estimation and valorization for energy. J. S. Tumuluru, InTechGoogle Scholar
  16. Huang GL, Anderson TD, Clubb RT (2014) Engineering microbial surfaces to degrade lignocellulosic biomass. Bioengineered 5(2):96–106. CrossRefPubMedGoogle Scholar
  17. Hyeon JE, Shin SK, Han SO (2016) Design of nanoscale enzyme complexes based on various scaffolding materials for biomass conversion and immobilization. Biotechnol J 11(11):1386–1396. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Hyyrylainen HL, Bolhuis A, Darmon E, Muukkonen L, Koski P, Vitikainen M, Sarvas M, Pragai Z, Bron S, van Dijl JM, Kontinen VP (2001) A novel two-component regulatory system in Bacillus subtilis for the survival of severe secretion stress. Mol Microbiol 41(5):1159–1172. CrossRefPubMedGoogle Scholar
  19. Jensen CL, Stephenson K, Jorgensen ST, Harwood C (2000) Cell-associated degradation affects the yield of secreted engineered and heterologous proteins in the Bacillus subtilis expression system. Microbiology 146(10):2583–2594. CrossRefPubMedGoogle Scholar
  20. Jolliffe LK, Doyle RJ, Streips UN (1980) Extracellular proteases modify cell wall turnover in Bacillus subtilis. J Bacteriol 141(3):1199–1208PubMedPubMedCentralGoogle Scholar
  21. Jolliffe LK, Doyle RJ, Streips UN (1981) The energized membrane and cellular autolysis in Bacillus subtilis. Cell 25(3):753–763. CrossRefPubMedGoogle Scholar
  22. Kobayashi G, Toida J, Akamatsu T, Yamamoto H, Shida T, Sekiguchi J (2000) Accumulation of an artificial cell wall-binding lipase by Bacillus subtilis wprA and/or sigD mutants. FEMS Microbiol Lett 188(2):165–169. CrossRefPubMedGoogle Scholar
  23. Krishnappa L, Dreisbach A, Otto A, Goosens VJ, Cranenburgh RM, Harwood CR, Becher D, van Dijl JM (2013) Extracytoplasmic proteases determining the cleavage and release of secreted proteins, lipoproteins, and membrane proteins in Bacillus subtilis. J Proteome Res 12(9):4101–4110. CrossRefPubMedGoogle Scholar
  24. Ledeaux JR, Grossman AD (1995) Isolation and characterization of kinC, a gene that encodes a sensor kinase homologous to the sporulation sensor kinases kinA and kinB in Bacillus subtilis. J Bacteriol 177(1):166–175. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Lee SY, Choi JH, Xu Z (2003) Microbial cell-surface display. Trends Biotechnol 21(1):45–52CrossRefPubMedGoogle Scholar
  26. Li PS, Tao HC (2015) Cell surface engineering of microorganisms towards adsorption of heavy metals. Crit Rev Microbiol 41(2):140–149. CrossRefPubMedGoogle Scholar
  27. Liew PX, Wang CL, Wong SL (2012) Functional characterization and localization of a Bacillus subtilis sortase and its substrate and use of this sortase system to covalently anchor a heterologous protein to the B. subtilis cell wall for surface display. J Bacteriol 194(1):161–175. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Link AJ, LaBaer J (2011) Trichloroacetic acid (TCA) precipitation of proteins. Cold Spring Harb Protoc 2011(8):993–994. CrossRefPubMedGoogle Scholar
  29. Liu L, Liu YF, Shin HD, Chen RR, Wang NS, Li JH, Du GC, Chen J (2013) Developing Bacillus spp. as a cell factory for production of microbial enzymes and industrially important biochemicals in the context of systems and synthetic biology. Appl Microbiol Biotechnol 97(14):6113–6127. CrossRefPubMedGoogle Scholar
  30. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 25(4):402–408. CrossRefPubMedGoogle Scholar
  31. Marciniak BC, Trip H, van-der Veek PJ, Kuipers OP (2012) Comparative transcriptional analysis of Bacillus subtilis cells overproducing either secreted proteins, lipoproteins or membrane proteins. Microb Cell Factories 11:66. CrossRefGoogle Scholar
  32. Mascher T, Zimmer SL, Smith TA, Helmann JD (2004) Antibiotic-inducible promoter regulated by the cell envelope stress-sensing two-component system LiaRS of Bacillus subtilis. Antimicrob Agents Chemother 48(8):2888–2896. CrossRefPubMedPubMedCentralGoogle Scholar
  33. McGivney E, Han LC, Avellan A, VanBriesen J, Gregory KB (2017) Disruption of autolysis in Bacillus subtilis using TiO2 nanoparticles. Sci Rep 7 doi:
  34. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428. CrossRefGoogle Scholar
  35. Mohamad NR, Marzuki NHC, Buang NA, Huyop F, Wahab RA (2015) An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Biotechnol Biotechnol Equip 29(2):205–220. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Nguyen HD, Schumann W (2006) Establishment of an experimental system allowing immobilization of proteins on the surface of Bacillus subtilis cells. J Biotechnol 122(4):473–482. CrossRefPubMedGoogle Scholar
  37. Nguyen HD, Phan TT, Schumann W (2011) Analysis and application of Bacillus subtilis sortases to anchor recombinant proteins on the cell wall. AMB Express 1(1):22. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Noone D, Howell A, Collery R, Devine KM (2001) YkdA and YvtA, HtrA-like serine proteases in Bacillus subtilis, engage in negative autoregulation and reciprocal cross-regulation of ykdA and yvtA gene expression. J Bacteriol 183(2):654–663. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Olson DG, McBride JE, Joe Shaw A, Lynd LR (2012) Recent progress in consolidated bioprocessing. Curr Opin Biotechnol 23(3):396–405. CrossRefPubMedGoogle Scholar
  40. Petre J, Longin R, Millet J (1981) Purification and properties of an endo-beta-1,4-glucanase from Clostridium thermocellum. Biochimie 63(7):629–639. CrossRefPubMedGoogle Scholar
  41. Pohl S, Harwood CR (2010) Heterologous protein secretion by Bacillus species from the cradle to the grave. Adv Appl Microbiol 73:1–25. CrossRefPubMedGoogle Scholar
  42. Pohl S, Bhavsar G, Hulme J, Bloor AE, Misirli G, Leckenby MW, Radford DS, Smith W, Wipat A, Williamson ED, Harwood CR, Cranenburgh RM (2013) Proteomic analysis of Bacillus subtilis strains engineered for improved production of heterologous proteins. Proteomics 13(22):3298–3308. CrossRefPubMedGoogle Scholar
  43. Rice KC, Bayles KW (2008) Molecular control of bacterial death and lysis. Microbiol Mol Biol Rev 72(1):85–109. CrossRefPubMedPubMedCentralGoogle Scholar
  44. Salzberg LI, Helmann JD (2007) An antibiotic-inducible cell wall-associated protein that protects Bacillus subtilis from autolysis. J Bacteriol 189(13):4671–4680. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Sarvas M, Harwood CR, Bron S, van Dijl JM (2004) Post-translocational folding of secretory proteins in Gram-positive bacteria. Biochim Biophys Acta 1694(1–3):311–327. PubMedCrossRefGoogle Scholar
  46. Schallmey M, Singh A, Ward OP (2004) Developments in the use of Bacillus species for industrial production. Can J Microbiol 50(1):1–17. CrossRefPubMedGoogle Scholar
  47. Schuurmann J, Quehl P, Festel G, Jose J (2014) Bacterial whole-cell biocatalysts by surface display of enzymes: toward industrial application. Appl Microbiol Biotechnol 98(19):8031–8046. CrossRefPubMedGoogle Scholar
  48. Schwarz WH, Grabnitz F, Staudenbauer WL (1986) Properties of a Clostridium thermocellum endoglucanase produced in Escherichia coli. Appl Environ Microbiol 51(6):1293–1299PubMedPubMedCentralGoogle Scholar
  49. Sirisha VL, Jain A, Jain A (2016) Enzyme immobilization: an overview on methods, support material, and applications of immobilized enzymes. Adv Food Nutr Res 79:179–211. CrossRefPubMedGoogle Scholar
  50. Smith TJ, Blackman SA, Foster SJ (2000) Autolysins of Bacillus subtilis: multiple enzymes with multiple functions. Microbiology 146:249–262. CrossRefPubMedGoogle Scholar
  51. Smith MR, Khera E, Wen F (2015) Engineering novel and improved biocatalysts by cell surface display. Ind Eng Chem Res 54(16):4021–4032. CrossRefPubMedPubMedCentralGoogle Scholar
  52. Spirig T, Weiner EM, Clubb RT (2011) Sortase enzymes in Gram-positive bacteria. Mol Microbiol 82(5):1044–1059. CrossRefPubMedPubMedCentralGoogle Scholar
  53. Stephenson K, Harwood CR (1998) Influence of a cell-wall-associated protease on production of alpha-amylase by Bacillus subtilis. Appl Environ Microbiol 64(8):2875–2881PubMedPubMedCentralGoogle Scholar
  54. Stephenson K, Bron S, Harwood CR (1999) Cellular lysis in Bacillus subtilis; the affect of multiple extracellular protease deficiencies. Lett Appl Microbiol 29(2):141–145CrossRefGoogle Scholar
  55. Strauss A, Gotz F (1996) In vivo immobilization of enzymatically active polypeptides on the cell surface of Staphylococcus carnosus. Mol Microbiol 21(3):491–500. CrossRefPubMedGoogle Scholar
  56. Tjalsma H, Antelmann H, Jongbloed JD, Braun PG, Darmon E, Dorenbos R, Dubois JY, Westers H, Zanen G, Quax WJ, Kuipers OP, Bron S, Hecker M, van Dijl JM (2004) Proteomics of protein secretion by Bacillus subtilis: separating the “secrets” of the secretome. Microbiol Mol Biol Rev 68(2):207–233. CrossRefPubMedPubMedCentralGoogle Scholar
  57. Wang LF, Bruckner R, Doi RH (1989) Construction of a Bacillus subtilis mutant-deficient in 3 extracellular proteases. J Gen Appl Microbiol 35(6):487–492CrossRefGoogle Scholar
  58. Wernerus H, Stahl S (2004) Biotechnological applications for surface-engineered bacteria. Biotechnol Appl Biochem 40(3):209–228. CrossRefPubMedGoogle Scholar
  59. Westers L, Westers H, Quax WJ (2004) Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism. Biochim Biophys Acta 1694(1–3):299–310. CrossRefPubMedGoogle Scholar
  60. Westers H, Westers L, Darmon E, van Dijl JM, Quax WJ, Zanen G (2006) The CssRS two-component regulatory system controls a general secretion stress response in Bacillus subtilis. FEBS J 273(16):3816–3827. CrossRefPubMedGoogle Scholar
  61. Westers L, Westers H, Zanen G, Antelmann H, Hecker M, Noone D, Devine KM, van Dijl JM, Quax WJ (2008) Genetic or chemical protease inhibition causes significant changes in the Bacillus subtilis exoproteome. Proteomics 8(13):2704–2713. CrossRefPubMedGoogle Scholar
  62. Wolf D, Kalamorz F, Wecke T, Juszczak A, Mader U, Homuth G, Jordan S, Kirstein J, Hoppert M, Voigt B, Hecker M, Mascher T (2010) In-depth profiling of the LiaR response of Bacillus subtilis. J Bacteriol 192(18):4680–4693. CrossRefPubMedPubMedCentralGoogle Scholar
  63. Wu SC, Yeung JC, Duan Y, Ye R, Szarka SJ, Habibi HR, Wong SL (2002) Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis: effects of molecular chaperones and a wall-bound protease on antibody fragment production. Appl Environ Microbiol 68(7):3261–3269. CrossRefPubMedPubMedCentralGoogle Scholar
  64. Ye R, Yang L, Wong S (1996) Construction of protease-deficient Bacillus subtilis strains for expression studies: inactivation of seven extracellular protease and the intracellular LonA protease. Proceedings of the International Symposium on Recent Advances in Bioindustry. The Korean Society for Applied Microbiology, Seoul, pp 160–169Google Scholar
  65. You C, Zhang XZ, Sathitsuksanoh N, Lynd LR, Zhang YH (2012) Enhanced microbial utilization of recalcitrant cellulose by an ex vivo cellulosome-microbe complex. Appl Environ Microbiol 78(5):1437–1444. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryUniversity of California, Los AngelesLos AngelesUSA
  2. 2.UCLA-DOE Institute of Genomics and ProteomicsUniversity of California, Los AngelesLos AngelesUSA
  3. 3.Molecular Biology InstituteUniversity of California, Los AngelesLos AngelesUSA
  4. 4.Department of Biological ChemistryUniversity of California, Los AngelesLos AngelesUSA

Personalised recommendations