A Bacterial Machinery for Surface Displayed Enzymes

Year 2018, Volume: 46 Issue: 2, 307 - 313, 03.06.2018

Urartu Özgür Şafak Şeker

Abstract

Biomaterial based protein delivery systems have been utilized for many applications in biomedicine. Despite
their great success, there is a need to develop innovative living, decision making systems for protein
delivery. In this context, here, a cellular system is proposed for protein release and delivery. Such systems can
be used not for biomedical purposes but also for other biochemical applications. In this regard a Escherichia
coli autotransporter protein, Ag43 was engineered to display on its cell membrane. Using this system alkaline
phosphatase protein is displayed on the cell surface as a fusion of Ag43-ALP which is also carrying a specific
TEV protease excision site. It was shown that the active from of the enzyme was released upon its interaction
with TEV protease from the cell surface. In this study a cellular machinery is proposed to be used as a controlled
enzyme delivery system. 

Keywords

Whole cell biocatalysis, autotransporter, synthetic biology

References

• D.D. Boehr, R.N. D’Amico, N. Rebecca, K.F. O’Rourke, F. Kathleen, Engineered control of enzyme structural dynamics and function, Prot. Sci., 27 (2018) 825-838.
• C. Silva, M. Martins, S. Jing, J.J. Fu, J. Fu, A. CavacoPaulo, Practical insights on enzyme stabilization, Crit. Rev. Biotechnol., 38 (2018) 335-350.
• J.H. Schrittwieser, S. Velikogne, M. Hall, Artificial biocatalytic linear cascades for preparation of organic molecules, Chem. Rev., 118 (2018) 270-348. Figure 5. ALP release from the surface of the cells displaying the enzyme upon addition of the TEV protease. The increase in the enzyme activity is statistically significant (t-test, p < 0.05). U.Ö.Ş. Şeker / Hacettepe J. Biol. & Chem., 2018, 46 (2), 307-313 313
• F. Kazenwadel; M. Franzreb, B.E. Rapp, Synthetic enzyme supercomplexes: co-immobilization of enzyme cascades, Anal. Methods, 7 (2015) 4030- 4037.
• M. Jeschek, S. Panke, T.R., Artificial metalloenzymes on the verge of new-to-nature metabolism, Trends Biotech. 36 (2018) 60-72.
• T. Nicolay, J. Vanderleyden, S. Spaepen, Autotransporter-based cell surface display in gramnegative bacteria, Critical Reviews in Microbiology, 41 (2015) 109-123.
• H. Nakatani, K. Hori, Cell surface protein engineering for high-performance whole-cell catalysts, Frontiers of Chemical Science and Engineering, 1 (2017) 46-57.
• Decorating microbes: surface display of proteins on Escherichia coli, E. van Bloois, R.T. Winter, H. Kolmar, M.W. Fraaije, Trends in Biotechnology, 29 (2011) 79- 86.
• N. Dautin, H. D. Bernstein, Protein secretion in gramnegative bacteria via the autotransporter pathway. Annu. Rev. Microbiol., 61 (2007) 89–112.
• W. Haagmans, M. van der Woude M., Phase variation of Ag43 in Escherichia coli: Dam-dependent methylation abrogates OxyR binding and OxyRmediated repression of transcription, Mol. Microbiol., 35 (2000) 877–887.
• D.E. Waldron, P. Owen, C.J. Dorman, Competitive interaction of the OxyR DNA-binding protein and the Dam methylase at the antigen 43 gene regulatory region in Escherichia coli, Mol. Microbiol., 44 (2002) 509–520.
• A. Wallecha, V. Munster, J. Correnti, T. Chan, M. van der Woude, Dam- and OxyR-dependent phase variation of agn43: essential elements and evidence for a new role of DNA methylation, J. Bacteriol., 184 (2002) 3338–3347.
• G.C. Ulett, J. Valle, C. Beloin, O. Sherlock, J.M. Ghigo, M. A. Schembri1, Functional analysis of antigen 43 in uropathogenic Escherichia coli reveals a role in longterm persistence in the urinary tract, Infect. Immun., 75 (2007) 3233-3244.
• H. Hasman, T. Chakraborty, P. J. Klemm, Antigen43-mediated autoaggregation of Escherichia coli is blocked by fimbriation, J. Bacteriol., 181(1999) 4834- 4841.
• K. Kjaergaard, M.A. Schembri, H. Hasman, P.J. Klemm, Antigen 43 from Escherichia coli induces inter- and intraspecies cell aggregation and changes in colony morphology of Pseudomonas fluorescens, J. Bacteriol., 182 (2000) 4789-4796.
• O. Sherlock, U. Dobrindt, J.B. Jensen, R.V. Munk, P. Klemm, Glycosylation of the self-recognizing Escherichia coli Ag43 autotransporter protein, J. Bacteriol., 188 (2006) 1789-1807.
• K. Kjaergaard, H. Hasman, M.A. Schembri, P. Klemm, Antigen 43-mediated autotransporter display, a versatile bacterial cell surface presentation system, J. Bacteriol., 184 (2002) 4197-4204.
• F.Y. Huang, C.C. Wang; Y.H. Huang, H.G. Zhao, J.L. Guo, S.L. Zhou, H. Wang, Y.Y. Lin, G.H. Tan, Antigen 43/Fc epsilon 3 chimeric protein expressed by a novel bacterial surface expression system as an effective asthma vaccine, Immunology, 143 (2014) 230-240.
• I. Munoz-Gutierrez, C. Moss-Acosta, B. Trujillo-Martinez, G. Gosset, A. Martinez, Ag43-mediated display of a thermostable beta-glucosidase in Escherichia coli and its use for simultaneous saccharification and fermentation at high temperatures, Microb. Cell Fact., 13 (2014) 106- .
• D.G. Gibson, L. Young, R.Y. Chuang, J.C. Venter, C.A. Hutchison, H.O. Smith, Enzymatic assembly of DNA molecules up to several hundred kilobases, Nat. Methods, 6 (2009) 343-345.
• C.W. Lee, S.H. Jang, H. Chung, Improving the stability of cold-adapted enzymes by immobilization, Catalysts, 7 (2017) 112.
• Y. Liu, D.S. Kim, M. Jewett, C. Michael, Repurposing ribosomes for synthetic biology, Curr. Opin. Chem. Biol., 40 (2017) 87-94.
• R. Lutz, H. Bujard, Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I-1-I-2 regulatory elements, Nucleic Acids Res., 25 (1997) 1203-1210.
• C. Magni, F. Sessa, J. Capraro, M. Duranti, E. Maffioli, A. Scarafoni, Structural and functional insights into the basic globulin 7S of soybean seeds by using trypsin as a molecular probe, Biochem. Biophys. Res. Commun., 496 (2018) 89-94.