VP (versatile peroxidases, EC 1.11.1.16) secreted by white‐rot fungi are involved in natural decay of lignin. VP combine the general catalytic features of other haem‐containing enzymes (in terms of substrate specificity and reaction mechanisms), such as the high‐redoxpotential ligninolytic peroxidases, LiP (lignin peroxidase) and MnP (manganese peroxidase), with those of peroxidases with a lower redox potential, such as HRP (horseradish peroxidase) and CiP (Coprinopsis cinerea peroxidase). Thus VP behaves as a generalist biocatalyst, readily oxidizing a variety of compounds. Unfortunately, VP has not been successfully functional expressed in any heterologous host, which limits its potential development. In this context, directed molecular evolution represents an elegant shortcut to tailor enzymes with improved features. By mimicking the Darwinist algorithm of natural selection through iterative steps of random mutagenesis and/or DNA recombination, the temporal scale of evolution can be collapsed from millions of years into months rather than weeks of bench work. We have engineered the VP from Pleurotus eryngii to be functionally expressed in Saccharomyces cerevisiae by directed evolution. Firstly, the optimization of culture conditions for functional expression and the engineering of a reliable high‐throughput screening assay were performed. Afterwards, a fusion gene containing the VP from P. eryngii and the α factor preproleader from S. cerevisiae was constructed and subjected to four rounds of directed evolution, achieving a level of secretion in S. cerevisiae of 21 mg/L. The evolved variant for expression (R4) harbored four mutations and increased its total VP activity 129‐fold over parent type along with a noticeable improvement of the catalytic efficiency at the haem channel oxidation site. Whilst the catalytic Trp was unaltered after evolution, the Mn2+ oxidation site was negatively affected by the mutations. The signal leader processing by the STE13 protease at the Golgi compartment changed as consequence of VP expression, retaining the additional N‐terminal sequence EAEA (Glu‐Ala‐Glu‐Ala) that enhanced secretion. The engineered N‐terminally truncated variants displayed similar biochemical properties to those of the non‐truncated counterpart in terms of kinetics, stability and spectroscopic features. Finally, we took advantage of the laboratory evolution platform set here to improve the thermostability of VP. Three additional cycles of evolution led to a more thermostable variant (2‐1B), harboring 3 stabilizing mutations. 2‐1B mutant showed a T50 8°C higher than parental type and the thermoactivity range was widened (from 30‐45°C for parent type to 30‐50°C for 2‐ 1B). Moreover, as a consequence of laboratory evolution, some unexpected side‐effects were detected. The enzyme’s stability at alkaline pHs was significantly increased retaining ~60 % of its residual activity at pH 9.0. In addition, the Km for H2O2 was enhanced up to 15‐fold while the catalytic efficiency was maintained. Mutations introduced in the course of evolution seemed to affect secretion, stability and activities by establishing new interactions with surrounding residues.

Proyectos europeos (Ref. BIORENEW, NMP2‐CT‐2006‐026456), (Ref. 3D‐NANOBIODEVICE, NMP4‐SL‐2009‐229255) , (Ref PEROXICATS, FP7‐KBBE‐2010‐4) y (Ref. CASCAT, CM0701) y Proyecto del Plan Nacional (EVOFACEL, Ref: BIO2010‐19697)

208 p.-75 fig.-42 tab.-2 anexos.

Peer reviewed