Figure 1.
General approach for MORPHING.
Segments subjected to random mutagenesis (M1 to M4) are shown in color and the non-mutagenized high fidelity amplified segments (HF1 to HF5) in light grey. After PCR, the pool of segments is co-transformed into S. cerevisiae along with the linearized vector. Overlapping areas of ∼50 bp flanking each segment allow specific crossover events to occur between fragments (represented by crosses), giving rise to an autonomously repaired vector carrying a full version of the target gene with random mutations (yellow stars) only in the defined regions.
Figure 2.
Overview of Versatile Peroxidase from Pleurotus eryngii (PDB ID: 3FJW).
Heme prostetic group and catalytic residues are shown as sticks in CPK color. The three catalytic sites are shown in detail. (A) The Mn2+ binding site (Mn2+, black sphere), the distal and proximal His (His47 and His169) the Arg43 involved in H2O2 reduction and the coordinating triad (Glu36, Glu40, Asp175) are depicted. (B) Heme access channel represented as electrostatics surface with heme group (stick mode) at the bottom of channel. (C) Catalytic Trp164 (LRET; long range electron transfer).
Figure 3.
Structural alignment for oxidative stability.
(A, B) Front and upper view (cartoon) with the protein surfaces shown in white. (C) Structural alignment in ribbon mode. The prosthetic heme group is highlighted in CPK colors. (D) Identities and similarities for each protein scaffold using the following PDB entries for the analysis: 1W4W, horseradish peroxidase C1A (HRP) expressed in E. coli (green); 1IYN, chloroplastic ascorbate peroxidase (ApX) from Nicotiana tabacum expressed in E. coli (blue); 3M5Q, manganese peroxidase isozyme 1 (MnP) from Phanerochaete chrysosporium (yellow); 1H3J, peroxidase from Coprinopsis cinerea CiP (gray); and 3FJW, Versatile peroxidase (VPL2) from Pleurotus eryngii (red).
Figure 4.
Mutagenic landscapes of the distal His environment (L28-G57) generated using 0.92 ng/µL of DNA template, and 0.01 mM (black), 0.1 mM (dark gray), 0.2 mM (medium gray), 0.4 mM (light gray) of MnCl2, respectively.
The percentages indicate the number of clones having less than 10% of the parent enzyme's activity; the solid horizontal line indicating the parental activity in the assay.
Figure 5.
Mutational loads, PCR conditions and selected variants used for MORPHING of the distal His environment.
Point mutations are highlighted in red and the overhangs in black. Improvements in oxidative stability are indicated as the fold increase with respect to the parental type at a [H2O2]/[Enzyme] ratio of 3,000∶1. Sequence alignments with stable peroxidase scaffolds are depicted, framing the mutations at positions 40 and 45 for VP and 53 and 48 for CiP, respectively. The MSA (multiple sequence alignment) was generated with T-coffee software: http://www.igs.cnrs-mrs.fr/Tcoffee/tcoffee_cgi/index.cgi.
Figure 6.
(A) Apparent t1/2 vs H2O2 for the R4 parental type, 5A9 mutant (P141A), 1C12 mutant (T45A) and 3G10 mutant (E40K) in the presence of 3,000 equivalents of H2O2.
Horizontal line indicates 50% of residual activity. (B) T50 profiles (kinetic thermostability) of VP variants. White squares, R4 parental type; white circle, 5A9 mutant (P141A); black squares, 3G10 mutant (E40K); black circles, 1C12 mutant (T45A). Each point represents the mean and standard deviation of three independent experiments.
Figure 7.
Mutagenic landscapes for MORPHING of the signal peptide of UPO using ABTS (A) and NBD (B) in colorimetric assays.
Directed evolution landscapes of the whole UPO gene obtained with ABTS (C) and NBD (D) assays. The activity of the clones is plotted in descending order. The solid horizontal line indicates the activity of the parental type in the assay.