Laboratory Evolution of High-Redox Potential Laccases

Abstract

Thermostable laccases with a high-redox potential have been engineered through a strategy that combines directed evolution with rational approaches. The original laccase signal sequence was replaced by the α-factor prepro-leader, and the corresponding fusion gene was targeted for joint laboratory evolution with the aim of improving kinetics and secretion by Saccharomyces cerevisiae, while retaining high thermostability. After eight rounds of molecular evolution, the total laccase activity was enhanced 34,000-fold culminating in the OB-1 mutant as the last variant of the evolution process, a highly active and stable enzyme in terms of temperature, pH range, and organic cosolvents. Mutations in the hydrophobic core of the evolved α-factor prepro-leader enhanced functional expression, whereas some mutations in the mature protein improved its catalytic capacities by altering the interactions with the surrounding residues.

Accurate Stabilities of Laccase Mutants Predicted with a Modified FoldX Protocol

“…The recent thermostability data for the WT, three single-point mutants, and one double mutant of the Trametes versicolor laccase TvLIIIb (UniProt ID: Q96UT7) 36 and nine multisite mutants 37 of the laccase PM1L (UniProt ID: Q12571) 35 were used for benchmarking. The structure of TvLIIIb has been solved by X-ray diffraction (PDB-ID: 1KYA), 40 and PM1L was homologous enough with the Trametes trogii laccase (PDB-ID: 2HRG) to produce a highquality homology model, enabling the structural input for FoldX.…”

“…The TvLIIIb data set consists mostly of single-point mutants, 36 whereas the PM1L data set contains nine multisite mutants. 37 TvLIIIb and PM1L share 80% sequence identity, and both basidomycete organisms have been associated with ligninolytic processes. TvLIIIb studied in this work is the most thermostable (T opt = 80°C, pH opt = 2.3 with ABTS as substrate) of four isoforms isolated from Trametes versicolor.…”

“…All copper ions were assigned a charge of +1, and the T2/T3 oxygen species were modeled as neutral water molecules, consistent with the X-ray information and preserving a realistic charge state of the protein. While exact changes in redox, protonation, and charge states are essentially impossible to model with classical force fields, for the purposes here, we were interested in the contribution to overall stability of amino acid side chains not directly interacting with copper (at distances >8 Å from any copper site 37 ), making this study less sensitive to state-specific issues than studies aiming at understanding metal-site properties.…”

“…39 It resembles other HRPLs from Trametes C30 (99% sequence identity), Trametes trogii (97% sequence identity), and Coriolopsis gallica (96% sequence identity). PM1L also has T opt = 80°C with guaiacol as a substrate, 39 is stable at pH 3−9, and has a standard reduction potential in excess of 700 mV, 37 making it of interest in biotechnological applications. The TvL mutants were generated by site-directed mutagenesis to improve oxidation of bulky phenolic substrates, 36 while the PM1L mutants resulted from a strategy combining directed evolution with rational approaches to facilitate yeast expression of highly stable and active HPRL mutants.…”

“…The latter approach culminated in the OB-1 mutant, which possessed a very high k cat /K M for substrates such as ABTS while preserving a substantial stability at elevated T, diverse pH, and organic cosolvents. 37 These promising laccases thus possess both high redox potentials and at the same time exquisite thermostability.…”