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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. Clubb
Applied genetics and molecular biotechnology

Abstract

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.

Keywords

Bacillus subtilis Protease-deficient Cell surface display LysM 

Notes

Acknowledgements

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)

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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

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