Accelerated directed evolution of dye-decolorizing peroxidase using a bacterial extracellular protein secretion system (BENNY)

Abstract

Background

Dye-decolorizing peroxidases (DyPs) are haem-containing peroxidases that show great promises in industrial biocatalysis and lignocellulosic degradation. Through the use of Escherichia coli osmotically-inducible protein Y (OsmY) as a bacterial extracellular protein secretion system (BENNY), we successfully developed a streamlined directed evolution workflow to accelerate the protein engineering of DyP4 from Pleurotus ostreatus strain PC15.

Result

After 3 rounds of random mutagenesis with error-prone polymerase chain reaction (epPCR) and 1 round of saturation mutagenesis, we obtained 4D4 variant (I56V, K109R, N227S and N312S) that displays multiple desirable phenotypes, including higher protein yield and secretion, higher specific activity (2.7-fold improvement in k_cat/K_m) and higher H₂O₂ tolerance (sevenfold improvement based on IC₅₀).

Conclusion

To our best knowledge, this is the first report of applying OsmY to simplify the directed evolution workflow and to direct the extracellular secretion of a haem protein such as DyP4.

Keywords

Directed evolution / Extracellular protein secretion / Dye-decolorizing peroxidase / Osmotically-inducible protein Y / Hydrogen peroxide tolerance

References

[1] Brissos V, Tavares D, Sousa AC, Robalo MP, Martins LO. Engineering a bacterial DyP-type peroxidase for enhanced oxidation of lignin-related phenolics at alkaline pH. ACS Catal, 2017, 7: 3454-3465.

[2] Camarero S, Galletti GC, Martinez AT. Preferential degradation of phenolic lignin units by two white rot fungi. Appl Environ Microbiol, 1994, 60: 4509-4516.

[3] Cheng CM, Tzou SC, Zhuang YH, Huang CC, Kao CH, Liao KW, Cheng TC, Chuang CH, Hsieh YC, Tai MH, Cheng TL. Functional production of a soluble and secreted single-chain antibody by a bacterial secretion system. PLoS ONE, 2014, 9: e97367.

[4] Fawal N, Li Q, Savelli B, Brette M, Passaia G, Fabre M, Mathe C, Dunand C. PeroxiBase: a database for large-scale evolutionary analysis of peroxidases. Nucleic Acids Res, 2013, 41: D441-444.

[5] Fernandez-Fueyo E, Linde D, Almendral D, Lopez-Lucendo MF, Ruiz-Duenas FJ, Martinez AT. Description of the first fungal dye-decolorizing peroxidase oxidizing manganese (II). Appl Microbiol Biotechnol, 2015, 99: 8927-8942.

[6] Gupta S, Adlakha N, Yazdani SS. Efficient extracellular secretion of an endoglucanase and a beta-glucosidase in E. coli. Protein Expr Purif, 2013, 88: 20-25.

[7] Hofrichter M, Ullrich R, Pecyna MJ, Liers C, Lundell T. New and classic families of secreted fungal heme peroxidases. Appl Microbiol Biotechnol, 2010, 87: 871-897.

[8] Kotzsch A, Vernet E, Hammarstrom M, Berthelsen J, Weigelt J, Graslund S, Sundstrom M. A secretory system for bacterial production of high-profile protein targets. Protein Sci, 2011, 20: 597-609.

[9] Le Y, Wang H. High-level soluble expression of a thermostable xylanase from thermophilic fungus Thermomyces lanuginosus in Escherichia coli via fusion with OsmY protein. Protein Expr Purif, 2014, 99: 1-5.

[10] Li S, Schoneich C, Borchardt RT. Chemical instability of protein pharmaceuticals: mechanisms of oxidation and strategies for stabilization. Biotechnol Bioeng, 1995, 48: 490-500.

[11] Martinez AT, Ruiz-Duenas FJ, Camarero S, Serrano A, Linde D, Lund H, Vind J, Tovborg M, Herold-Majumdar OM, Hofrichter M, Liers C, Ullrich R, Scheibner K, Sannia G, Piscitelli A, Pezzella C, Sener ME, Kilic S, van Berkel WJH, Guallar V, Lucas MF, Zuhse R, Ludwig R, Hollmann F, Fernandez-Fueyo E, Record E, Faulds CB, Tortajada M, Winckelmann I, Rasmussen JA, Gelo-Pujic M, Gutierrez A, Del Rio JC, Rencoret J, Alcalde M. Oxidoreductases on their way to industrial biotransformations. Biotechnol Adv, 2017, 35: 815-831.

[12] Natarajan A, Haitjema CH, Lee R, Boock JT, DeLisa MP. An engineered survival-selection assay for extracellular protein expression uncovers hypersecretory phenotypes in Escherichia coli. ACS Synth Biol, 2017, 6: 875-883.

[13] Ogola HJ, Kamiike T, Hashimoto N, Ashida H, Ishikawa T, Shibata H, Sawa Y. Molecular characterization of a novel peroxidase from the cyanobacterium Anabaena sp. strain PCC 7120. Appl Environ Microbiol, 2009, 75: 7509-7518.

[14] Ogola HJ, Hashimoto N, Miyabe S, Ashida H, Ishikawa T, Shibata H, Sawa Y. Enhancement of hydrogen peroxide stability of a novel Anabaena sp. DyP-type peroxidase by site-directed mutagenesis of methionine residues. Appl Microbiol Biotechnol, 2010, 87: 1727-1736.

[15] Otieno BA, Krause CE, Rusling JF. Bioconjugation of antibodies and enzyme labels onto magnetic beads. Methods Enzymol, 2016, 571: 135-150.

[16] Pardo I, Chanaga X, Vicente AI, Alcalde M, Camarero S. New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass. BMC Biotechnol, 2013, 13: 90.

[17] Qian ZG, Xia XX, Choi JH, Lee SY. Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in Escherichia coli. Biotechnol Bioeng, 2008, 101: 587-601.

[18] Ruano-Gallego D, Fraile S, Gutierrez C, Fernandez LA. Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system. Microb Cell Fact, 2019, 18: 47.

[19] Salvachua D, Prieto A, Martinez AT, Martinez MJ. Characterization of a novel dye-decolorizing peroxidase (DyP)-type enzyme from Irpex lacteus and its application in enzymatic hydrolysis of wheat straw. Appl Environ Microbiol, 2013, 79: 4316-4324.

[20] Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc, 2006, 1: 2856-2860.

[21] Singh R, Eltis LD. The multihued palette of dye-decolorizing peroxidases. Arch Biochem Biophys, 2015, 574: 56-65.

[22] Tee KL, Wong TS. Polishing the craft of genetic diversity creation in directed evolution. Biotechnol Adv, 2013, 31: 1707-1721.

[23] Warburton M, Omar Ali H, Liong WC, Othusitse AM, Abdullah Zubir AZ, Maddock S, Wong TS. OneClick: a program for designing focused mutagenesis experiments. AIMS Bioengineering, 2015, 2: 126-143.

[24] Wong TS, Zhurina D, Schwaneberg U. The diversity challenge in directed protein evolution. Comb Chem High Throughput Screen, 2006, 9: 271-288.

[25] Zhang G, Brokx S, Weiner JH. Extracellular accumulation of recombinant proteins fused to the carrier protein YebF in Escherichia coli. Nat Biotechnol, 2006, 24: 100-104.

[26] Zheng Z, Chen T, Zhao M, Wang Z, Zhao X. Engineering Escherichia coli for succinate production from hemicellulose via consolidated bioprocessing. Microb Cell Fact, 2012, 11: 37.

Funding

Engineering and Physical Sciences Research Council(EP/E036252/1)

State Key Laboratory of Bioreactor Engineering

The Leverhulme Trust

The University of Sheffield

Biotechnology and Biological Sciences Research Council(BB/R020183/1)

European Cooperation in Science and Technology(CM1303)

University of Tabuk