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

Abdulrahman H A Alessa; Kang Lan Tee; David Gonzalez-Perez; Hossam E M Omar Ali; Caroline A Evans; Alex Trevaskis; Jian-He Xu; Tuck Seng Wong

doi:10.1186/s40643-019-0255-7

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

Bioresour Bioprocess. 2019;6(1):20. doi: 10.1186/s40643-019-0255-7. Epub 2019 May 31.

Authors

Abdulrahman H A Alessa^#¹, Kang Lan Tee^#¹, David Gonzalez-Perez¹, Hossam E M Omar Ali¹, Caroline A Evans¹, Alex Trevaskis¹, Jian-He Xu², Tuck Seng Wong¹

Affiliations

¹ 1Department of Chemical & Biological Engineering and Advanced Biomanufacturing Centre, University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield, S1 3JD UK.
² 2Laboratory of Biocatalysis and Bioprocessing, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237 People's Republic of China.

^# Contributed equally.

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; Dye-decolorizing peroxidase; Extracellular protein secretion; Hydrogen peroxide tolerance; Osmotically-inducible protein Y.