Engineering of Unspecific Peroxygenases Using a Superfolder-Green-Fluorescent-Protein-Mediated Secretion System in Escherichia coli

Abstract

Unspecific peroxygenases (UPOs), secreted by fungi, demonstrate versatility in catalyzing challenging selective oxyfunctionalizations. However, the number of peroxygenases and corresponding variants with tailored selectivity for a broader substrate scope is still limited due to the lack of efficient engineering strategies. In this study, a new unspecific peroxygenase from Coprinopsis marcescibilis (CmaUPO) is identified and characterized. To enhance or reverse the enantioselectivity of wildtype (WT) CmaUPO catalyzed asymmetric hydroxylation of ethylbenzene, CmaUPO was engineered using an efficient superfolder-green-fluorescent-protein (sfGFP)-mediated secretion system in Escherichia coli. Iterative saturation mutagenesis (ISM) was used to target the residual sites lining the substrate tunnel, resulting in two variants: T125A/A129G and T125A/A129V/A247H/T244A/F243G. The two variants greatly improved the enantioselectivities [21% ee (R) for WT], generating the (R)-1-phenylethanol or (S)-1-phenylethanol as the main product with 99% ee (R) and 84% ee (S), respectively. The sfGFP-mediated secretion system in E. coli demonstrates applicability for different UPOs (AaeUPO, CciUPO, and PabUPO-I). Therefore, this developed system provides a robust platform for heterologous expression and enzyme engineering of UPOs, indicating great potential for their sustainable and efficient applications in various chemical transformations.

Figure 1

Bioinformatic workflow for the discovery of putative UPOs. Candidate sequences were mined using probes AaeUPO, CciUPO, and PabUPO. A maximum likelihood phylogenetic tree of the UPO candidate referenced the confirmed UPOs, bootstrap values at each node, were from 100 replicates. The probes are highlighted in red, and the UPO candidates selected are marked in blue.

Figure 2

CmaUPO substrate panel, with the oxidation sites highlighted. Reaction conditions: substrate (20 mM 1, 50 mM 2, 100 mM 3, 2.5 mM 4, and 10 mM 5), CmaUPO (0.15 μM for 1 and 2, 0.3 μM for 3, 1 μM for 4, and 1.5 μM for 5), 25 mM glucose, 100 U mL^–1 glucose oxidase, V_final = 1 mL, 30 °C, 220 rpm, 4 h. Additionally, 5 mM sodium ascorbate was added to substrate 2 to avoid phenoxy radical formation. All experiments were performed in triplicates. Product ratio was determined by the peak area or calibration with the commercial product standards on GC or HPLC.

Figure 3

Heterologous expression strategy of CmaUPO in E. coli. (A) Fusion-expression platform for CmaUPO production. CmaUPO was fused with sfGFP₁₅ in different plasmid configurations for heterologous expression in E. coli BL21 (DE3). Recombinant plasmids: (a) pET28a-CmaUPO; (b) pET28a-sfGFP₁₅-CmaUPO; (c) pET28a-CmaUPO-sfGFP₁₅; (d) pET23a-CmaUPO; (e) pET23a-sfGFP₁₅-CmaUPO; and (f) pET23a-CmaUPO-sfGFP₁₅. (B) Activity test of cell lysates of different recombinant E. coli cells expressing CmaUPO with ABTS and NBD. (C) Biotransformation catalyzed by the whole cells or lysate of different recombinant E. coli cells expressing CmaUPO with ethylbenzene as a substrate. Reaction conditions: ethylbenzene 10 mM, CmaUPO whole cell (OD₆₀₀ = 20, cell dry weight 8 g L^–1) or corresponding cell lysate, glucose 25 mM, glucose oxidase 100 U mL^–1, 100 mM potassium phosphate pH 7.5, V_final = 1 mL, 30 °C, 220 rpm, 4 h.

Figure 4

Structure-guided mutagenesis for improving the enantioselectivity of CmaUPO. (A) Analysis of protein tunnels using Caver 3.03. Two tunnels were identified as a substrate tunnel (displayed in blue sphere) and potential water/H₂O₂ transport channel (displayed in green mesh), respectively. (B) Zoom of the active sites (lining in the substrate tunnel) around substrate in 5 Å of CmaUPO. Within the substrate channel, the residual sites within a 5 Å radius around the substrate sites are divided into three groups. The first group includes the residual sites located at the entrance of the substrate channel (T125, F243, and F251), which are highlighted in yellow. The second group consists of the residual sites on the left side of the substrate in the second layer of the substrate channel (A129 and F173), marked with green. The third group comprises the residual sites on the right side of the substrate in the second layer of the substrate channel (T244 and A247), indicated by violet. Additionally, the substrate tunnel is visualized as a blue mesh. (C) The directed evolution of CmaUPO was carried out by ISM. The proportion of blue and orange squares represents the proportion of (R)-1-phenylethanol and (S)-1-phenylethanol products, respectively. (D) Typical GC chromatograms for CmaUPO variants catalyzed conversion of ethylbenzene. Reaction conditions: ethylbenzene 10 mM, CmaUPO whole cell, glucose 25 mM, glucose oxidase 100 U mL^–1, 100 mM potassium phosphate pH 7.5, V_final = 1 mL, 30 °C, 220 rpm, 10 min. All experiments were performed in triplicates.

Figure 5

Structural analysis and molecular docking of CmaUPO and its variants with ethylbenzene. (A) The surface structure of the active pocket inlet and active pocket volume diagram of WT CmaUPO. The volume of the active pocket is 373.01 ų. The volume was measured using the DoGSiteScorer tool of the ProteinsPlus platform (https://proteins.plus/). (B) The surface structure of the active pocket inlet and active pocket volume diagram of variant T125A. The volume of the active pocket is 500.80 ų. (C) Zoom of the active site of variant M2a (T125A/A129G). Key sites are marked in light blue sticks (target mutant residues marked in green) and labeled in black for mutant T125A/A129G. The substrate ethylbenzene is show in orange. (D) Zoom of the active site of variant M5 (T125A/A129V/A247H/T244A/F243G). Key sites are depicted using light blue sticks, with target mutant residues highlighted in green and labeled in black. The substrate ethylbenzene is represented in orange.

Figure 6

Versatility of the fusion-expression platform for other UPOs. (A) Lysate supernatant activities of UPOs measured with ABTS and NBD. (B) Asymmetric hydroxylation of ethylbenzene using recombinant E. coli cells expressing corresponding UPOs.