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

Fig. 1

Activity values (Abs₄₀₅) of OsmY-DyP4-catalysed ABTS oxidation in a 96-well plate, reported in a descending order (black—before background subtraction, blue—after background subtraction). The assay was conducted using a protocol streamlined with OsmY-based BENNY. The apparent coefficient of variance (CV) was calculated without subtracting the assay background, while the true CV was obtained after background subtraction

Fig. 2

a Products and side products of epPCRs with high (H), medium (M) and low (L) mutation rates. b ABTS oxidation activity values (Abs₄₀₅) of OsmY-DyP4 mutant libraries from the 3rd round of epPCR [blue—epPCR library with low (L) mutation rate, green—epPCR library with medium (M) mutation rate, red—epPCR library with high (H) mutation rate]. Solid and dotted horizontal lines represent the average activity of parental strain (i.e. 2A5) and one standard deviation (SD) above/below the average value, respectively

Fig. 3

Protein model of 4D4 variant, created with PyMOL using crystal structure of DyP4 F194Y variant (PDB 6FSK) as template. Missense mutations in 4D4 are indicated (I56V, K109R, N227S and N312S)

Fig. 4

UV-vis spectra of purified WT (black), 3F6 (blue) and 4D4 (red), indicating the presence of a Soret band at 406 nm with a slight shoulder at ~ 385 nm, Q band at 504 nm and a charge-transfer (CT) band at 637 nm

Fig. 5

Relative ABTS oxidation activities of purified WT (black), 3F6 (blue) and 4D4 (red), at increasing H₂O₂ concentrations from 0.15 to 50.00 mM, with ABTS concentration kept at 7 mM. The WT activity at 0.25 mM H₂O₂was arbitrarily set as 100% and used as a reference point

Fig. 6

Mass spectrometry data of SEC fractions F2 (a) and F7 (b) proteins. Mascot database searching against the Escherichia coli (strain B/BL21-DE3) reference proteome, plus the recombinant OsmY-DyP4 sequence, led to matching to this recombinant protein. Matched peptides are shown in red against the full-length amino acid sequence of the recombinant protein