Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

Abstract

c Unspecific peroxygenase (UPO) represents a new type of heme-thiolate enzyme with self-sufficient mono(per)oxygenase activity and many potential applications in organic synthesis. With a view to taking advantage of these properties, we subjected the Agrocybe aegerita UPO1-encoding gene to directed evolution in Saccharomyces cerevisiae. To promote functional expression, several different signal peptides were fused to the mature protein, and the resulting products were tested. Over 9,000 clones were screened using an ad hoc dual-colorimetric assay that assessed both peroxidative and oxygen transfer activities. After 5 generations of directed evolution combined with hybrid approaches, 9 mutations were introduced that resulted in a 3,250-fold total activity improvement with no alteration in protein stability. A breakdown between secretion and catalytic activity was performed by replacing the native signal peptide of the original parental type with that of the evolved mutant; the evolved leader increased functional expression 27-fold, whereas an 18-fold improvement in the k cat /K m value for oxygen transfer activity was obtained. The evolved UPO1 was active and highly stable in the presence of organic cosolvents. Mutations in the hydrophobic core of the signal peptide contributed to enhance functional expression up to 8 mg/liter, while catalytic efficiencies for peroxidative and oxygen transfer reactions were increased by several mutations in the vicinity of the heme access channel. Overall, the directed-evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships.

A Peroxygenase from Chaetomium globosum Catalyzes the Selective Oxygenation of Testosterone

“…Such an approach was recently described for an A. aegerita UPO that was expressed in S. cerevisiae and mutated in nine positions, and then optimized for recombinant protein production and secretion in P. pastoris 25, 55, 56. Moreover, modern protein engineering techniques might help to design tailor‐made UPOs for specific steroid hydroxylations and to overcome catalytic bottlenecks such as solvent and peroxide instability.…”

Synthesis of 1‐Naphthol by a Natural Peroxygenase Engineered by Directed Evolution

“…[23] The Phe residues involved in accommodating substrates at the catalytic pocket are highlighted in pink, the Cys residues are in light green (C36 axial ligand and the C278-C319 disulfide bridge), the R189 component of the acid-base pair involved in the catalysis is tagged in light blue and the Fe 3+ of heme is represented as a red sphere.…”

“…[23] PaDa-I harbors 9 mutations (F12Y-A14V-R15G-A21D-V57A-L67F-V75I-I248V-F311L: the mutations in the signal peptide are underlined) that enhance its functional expression in yeast (8 mg/L in S. cerevisiae and over 200 mg/L in Pichia pastoris). [24] At the same time this mutant retains strong activity and stability, particularly in terms of temperature and the presence of co-solvents.…”

“…The values were normalized against the parental type in the corresponding plate. To rule out false positives, two re-screenings were carried out and a third re-screening was performed in order to assess the kinetic stability (the protocol for the re-screenings and the studies of kinetic stability -T50-are described elsewhere [23] ).…”

“…[23] After each round of directed evolution, the PCR products were loaded onto a preparative agarose gel and purified using the Zymoclean Gel DNA Recovery kit. The recovered DNA fragments were cloned under the control of the GAL1 promoter of the pJRoC30 expression shuttle vector, using BamHI and XhoI to linearize the plasmid and remove the parental gene.…”