Skip to main content
SpringerLink
Log in

Display of Oligo-α-1,6-Glycosidase from Exiguobacterium sibiricum on the Surface of Escherichia coli Cells

  • Published:
Biochemistry (Moscow) Aims and scope Submit manuscript

Abstract

Cell-surface display using anchor motifs of outer membrane proteins allows exposure of target peptides and proteins on the surface of microbial cells. Previously, we obtained and characterized highly catalytically active recombinant oligo-α-1,6-glycosidase from the psychrotrophic bacterium Exiguobacterium sibiricum (EsOgl). It was also shown that the autotransporter AT877 from Psychrobacter cryohalolentis and its deletion variants efficiently displayed type III fibronectin (10Fn3) domain 10 on the surface of Escherichia coli cells. The aim of the work was to obtain an AT877-based system for displaying EsOgl on the surface of bacterial cells. The genes for the hybrid autotransporter EsOgl877 and its deletion mutants EsOgl877Δ239 and EsOgl877Δ310 were constructed, and the enzymatic activity of EsOgl877 was investigated. Cells expressing this protein retained ~90% of the enzyme maximum activity within a temperature range of 15-35°C. The activity of cells expressing EsOgl877Δ239 and EsOgl877Δ310 was 2.7 and 2.4 times higher, respectively, than of the cells expressing the full-size AT. Treatment of cells expressing EsOgl877 deletion variants with proteinase K showed that the passenger domain localized to the cell surface. These results can be used for further optimization of display systems expressing oligo-α-1,6-glycosidase and other heterologous proteins on the surface of E. coli cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.

Similar content being viewed by others

Abbreviations

AT:

autotransporter

EsOgl:

oligo-α-1,6-glycosidase from Exiguobacterium sibiricum

10Fn3:

human fibronectin type III domain 10

PBS:

phosphate buffered saline

pNPG:

p-nitrophenyl-α-D-glucopyranoside

References

  1. Van der Maarel, M. J. E. C., van der Veen, B., Uitdehaag, J. C. M., Leemhuis, H., and Dijkhuizen, L. (2002) Properties and applications of starch-converting enzymes of the α-amylase family, J. Biotechnol., 94, 137-155, https://doi.org/10.1016/s0168-1656(01)00407-2.

    Article  CAS  PubMed  Google Scholar 

  2. Hua, X., and Yang, R. (2016) Enzymes in starch processing, in Enzymes in Food and Beverage Processing (Chandrasekaran, M., ed.) CRC Press, Boca Raton, FL, USA, pp. 139-170.

  3. Dong, Z., Tang, C., Lu, Y., Yao, L., and Kan, Y. (2020) Microbial oligo‐α-1,6‐glucosidase: current developments and future perspectives, Starch Stärke, 72, 1900172, https://doi.org/10.1002/star.201900172.

    Article  CAS  Google Scholar 

  4. Watanabe, K., Hata, Y., Kizaki, H., Katsube, Y., and Suzuki, Y. (1997) The refined crystal structure of Bacillus cereus oligo-1,6-glucosidase at 2.0 Å resolution: structural characterization of proline-substitution sites for protein thermostabilization, J. Mol. Biol., 269, 142-153, https://doi.org/10.1006/jmbi.1997.1018.

    Article  CAS  PubMed  Google Scholar 

  5. Watanabe, K., Kitamura, K., and Suzuki, Y. (1996) Analysis of the critical sites for protein thermostabilization by proline substitution in oligo-1,6-glucosidase from Bacillus coagulans ATCC 7050 and the evolutionary consideration of proline residues, Appl. Environ. Microbiol., 62, 2066-2073, https://doi.org/10.1128/aem.62.6.2066-2073.1996.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Watanabe, K., Chishiro, K., Kitamura, K., and Suzuki, Y. (1991) Proline residues responsible for thermostability occur with high frequency in the loop regions of an extremely thermostable oligo-1,6-glucosidase from Bacillus thermoglucosidasius KP1006, J. Biol. Chem., 266, 24287-24294, https://doi.org/10.1016/S0021-9258(18)54226-5.

    Article  CAS  PubMed  Google Scholar 

  7. Feller, G. (2013) Psychrophilic enzymes: from folding to function and biotechnology, Scientifica, 2013, 512840, https://doi.org/10.1155/2013/512840.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Barroca, M., Santos, G., Gerday, C., and Collins, T. (2017) Biotechnological Aspects of Cold-Active Enzymes, in Psychrophiles: From Biodiversity to Biotechnology (Margesin, R., ed.) Springer International Publishing, Cham, pp. 461-475, https://doi.org/10.1007/978-3-319-57057-0_19.

  9. Berlina, Y. Y., Petrovskaya, L. E., Kryukova, E. A., Shingarova, L. N., Gapizov, S. S., Kryukova, M. V., Rivkina, E. M., Kirpichnikov, M. P., and Dolgikh, D. A. (2021) Engineering of Thermal stability in a cold-active oligo-1,6-glucosidase from Exiguobacterium sibiricum with unusual amino acid content, Biomolecules, 11, 1229, https://doi.org/10.3390/biom11081229.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Van Ulsen, P., ur Rahman, S., Jong, W. S., Daleke-Schermerhorn, M. H., and Luirink, J. (2014) Type V secretion: from biogenesis to biotechnology, Biochim. Biophys. Acta Mol. Cell Res., 1843, 1592-1611, https://doi.org/10.1016/j.bbamcr.2013.11.006.

    Article  CAS  Google Scholar 

  11. Nicolay, T., Vanderleyden, J., and Spaepen, S. (2015) Autotransporter-based cell surface display in Gram-negative bacteria, Crit. Rev. Microbiol., 41, 109-123, https://doi.org/10.3109/1040841X.2013.804032.

    Article  CAS  PubMed  Google Scholar 

  12. De Carvalho, C. C. (2017) Whole cell biocatalysts: essential workers from nature to the industry, Micr. Biotechnol., 10, 250-263, https://doi.org/10.1111/1751-7915.12363.

    Article  Google Scholar 

  13. Schüürmann, J., Quehl, P., Festel, G., and Jose, J. (2014) Bacterial whole-cell biocatalysts by surface display of enzymes: toward industrial application, Appl. Microbiol. Biotechnol., 98, 8031-8046, https://doi.org/10.1007/s00253-014-5897-y.

    Article  CAS  PubMed  Google Scholar 

  14. He, M.-X., Feng, H., and Zhang, Y.-Z. (2008) Construction of a novel cell-surface display system for heterologous gene expression in Escherichia coli by using an outer membrane protein of Zymomonas mobilis as anchor motif, Biotechnol. Lett., 30, 2111-2117, https://doi.org/10.1007/s10529-008-9813-3.

    Article  CAS  PubMed  Google Scholar 

  15. Ryu, S., and Karim, M. N. (2011) A whole cell biocatalyst for cellulosic ethanol production from dilute acid-pretreated corn stover hydrolyzates, Appl. Microbiol. Biotechnol., 91, 529-542, https://doi.org/10.1007/s00253-011-3261-z.

    Article  CAS  PubMed  Google Scholar 

  16. Muñoz-Gutiérrez, I., Oropeza, R., Gosset, G., and Martinez, A. (2012) Cell surface display of a β-glucosidase employing the type V secretion system on ethanologenic Escherichia coli for the fermentation of cellobiose to ethanol, J. Indust. Microbiol. Biotechnol., 39, 1141-1152, https://doi.org/10.1007/s10295-012-1122-0.

    Article  CAS  Google Scholar 

  17. Soma, Y., Inokuma, K., Tanaka, T., Ogino, C., Kondo, A., Okamoto, M., and Hanai, T. (2012) Direct isopropanol production from cellobiose by engineered Escherichia coli using a synthetic pathway and a cell surface display system, J. Biosci. Bioeng., 114, 80-85, https://doi.org/10.1016/j.jbiosc.2012.02.019.

    Article  CAS  PubMed  Google Scholar 

  18. Van Ulsen, P., Zinner, K. M., Jong, W. S. P., and Luirink, J. (2018) On display: autotransporter secretion and application, FEMS Microbiol. Lett., 365, fny165, https://doi.org/10.1093/femsle/fny165.

    Article  CAS  Google Scholar 

  19. Petrovskaya, L., Novototskaya-Vlasova, K., Kryukova, E., Rivkina, E., Dolgikh, D., and Kirpichnikov, M. (2015) Cell surface display of cold-active esterase EstPc with the use of a new autotransporter from Psychrobacter cryohalolentis K5T, Extremophiles, 19, 161-170, https://doi.org/10.1007/s00792-014-0695-0.

    Article  CAS  PubMed  Google Scholar 

  20. Petrovskaya, L., Zlobinov, A., Shingarova, L., Boldyreva, E., Gapizov, S. S., Novototskaya-Vlasova, K., Rivkina, E., Dolgikh, D., and Kirpichnikov, M. (2018) Fusion with the cold-active esterase facilitates autotransporter-based surface display of the 10th human fibronectin domain in Escherichia coli, Extremophiles, 22, 141-150, https://doi.org/10.1007/s00792-017-0990-7.

    Article  CAS  PubMed  Google Scholar 

  21. Shingarova, L., Petrovskaya, L., Zlobinov, A., Gapizov, S. S., Kryukova, E., Birikh, K., Boldyreva, E., Yakimov, S., Dolgikh, D., and Kirpichnikov, M. (2018) Construction of artificial TNF-binding proteins based on the 10th human fibronectin type III domain using bacterial display, Biochemistry (Moscow), 83, 708-716, https://doi.org/10.1134/S0006297918060081.

    Article  CAS  PubMed  Google Scholar 

  22. Shingarova, L. N., Petrovskaya, L. E., Kryukova, E. A., Gapizov, S. S., Boldyreva, E. F., Dolgikh, D. A., and Kirpichnikov, M. P. (2022) Deletion variants of autotransporter from Psychrobacter cryohalolentis increase efficiency of 10FN3 exposure on the surface of Escherichia coli cells, Biochemistry (Moscow), 87, 932-939, https://doi.org/10.1134/S0006297922090061.

    Article  CAS  PubMed  Google Scholar 

  23. Dalbey, R. E., and Kuhn, A. (2012) Protein traffic in Gram-negative bacteria – how exported and secreted proteins find their way, FEMS Microbiol. Rev., 36, 1023-1045, https://doi.org/10.1111/j.1574-6976.2012.00327.x.

    Article  CAS  PubMed  Google Scholar 

  24. Kim, K. H., Aulakh, S., and Paetzel, M. (2012) The bacterial outer membrane beta-barrel assembly machinery, Prot. Sci., 21, 751-768, https://doi.org/10.1002/pro.2069.

    Article  CAS  Google Scholar 

  25. Peterson, J. H., Tian, P., Ieva, R., Dautin, N., and Bernstein, H. D. (2010) Secretion of a bacterial virulence factor is driven by the folding of a C-terminal segment, Proc. Nat. Acad. Sci. USA, 107, 17739-17744, https://doi.org/10.1073/pnas.1009491107.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Junker, M., Besingi, R. N., and Clark, P. L. (2009) Vectorial transport and folding of an autotransporter virulence protein during outer membrane secretion, Mol. Microbiol., 71, 1323-1332, https://doi.org/10.1111/j.1365-2958.2009.06607.x.

    Article  CAS  PubMed  Google Scholar 

  27. Renn, J. P., Junker, M., Besingi, R. N., Braselmann, E., and Clark, P. L. (2012) ATP-independent control of autotransporter virulence protein transport via the folding properties of the secreted protein, Chem. Biol., 19, 287-296, https://doi.org/10.1016/j.chembiol.2011.11.009.

    Article  CAS  PubMed  Google Scholar 

  28. Braselmann, E., and Clark, P. L. (2012) Autotransporters: the Cellular environment reshapes a folding mechanism to promote protein transport, J. Phys. Chem. Lett., 3, 1063-1071, https://doi.org/10.1021/jz201654k.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Siddiqui, K. S., and Cavicchioli, R. (2006) Cold-adapted enzymes, Annu. Rev. Biochem., 75, 403-433, https://doi.org/10.1146/annurev.biochem.75.103004.142723.

    Article  CAS  PubMed  Google Scholar 

  30. Struvay, C., and Feller, G. (2012) Optimization to low temperature activity in psychrophilic enzymes, Int. J. Mol. Sci., 13, 11643-11665, https://doi.org/10.3390/ijms130911643.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Santiago, M., Ramírez-Sarmiento, C. A., Zamora, R. A., and Parra, L. P. (2016) Discovery, molecular mechanisms, and industrial applications of cold-active enzymes, Front. Microbiol., 7, https://doi.org/10.3389/fmicb.2016.01408.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Novototskaya-Vlasova, K., Petrovskaya, L., Yakimov, S., and Gilichinsky, D. (2012) Cloning, purification, and characterization of a cold adapted esterase produced by Psychrobacter cryohalolentis K5T from Siberian cryopeg, FEMS Microbiol. Ecol., 82, 367-375, https://doi.org/10.1111/j.1574-6941.2012.01385.x.

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

The work was performed as part of the State Assignment for the Institute of Bioorganic Chemistry of the Russian Academy of Sciences no. FMFM-2019-0007 (0101-2019-0007).

Author information

Authors and Affiliations

  1. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997, Moscow, Russia

    Lyudmila N. Shingarova, Lada E. Petrovskaya, Elena A. Kryukova, Sultan S. Gapizov, Dmitry A. Dolgikh & Mikhail P. Kirpichnikov

  2. Faculty of Biology, Lomonosov Moscow State University, 119234, Moscow, Russia

    Sultan S. Gapizov, Dmitry A. Dolgikh & Mikhail P. Kirpichnikov

Authors
  1. Lyudmila N. Shingarova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lada E. Petrovskaya
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Elena A. Kryukova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sultan S. Gapizov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dmitry A. Dolgikh
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mikhail P. Kirpichnikov
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.N.S., L.E.P., and M.P.K. developed the concept and supervised the study; L.N.S., E.A.K., and S.Sh.G.: performed the experiments; L.N.S., L.E.P., and D.A.D. discussed the results; L.E.P. and L.N.S. wrote the manuscript.

Corresponding author

Correspondence to Lyudmila N. Shingarova.

Ethics declarations

The authors declare no conflict of interest in financial or any other sphere. This article does not contain a description of studies with human participants or animals performed by any of the authors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shingarova, L.N., Petrovskaya, L.E., Kryukova, E.A. et al. Display of Oligo-α-1,6-Glycosidase from Exiguobacterium sibiricum on the Surface of Escherichia coli Cells. Biochemistry Moscow 88, 716–722 (2023). https://doi.org/10.1134/S0006297923050140

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0006297923050140

Keywords

Search

Navigation