Increasing Redox Potential, Redox Mediator Activity, and Stability in a Fungal Laccase by Computer-Guided Mutagenesis and Directed Evolution

Fungal high-redox-potential laccases (HRPLs) are multicopper oxidases with a relaxed substrate specificity that is highly dependent on their binding affinity and redox potential of the T1Cu site (ET1). In this study, we combined computational design with directed evolution to tailor an HRPL variant with increased ET1 and activity toward high-redox-potential mediators as well as enhanced stability. Laccase mutant libraries were screened in vitro using synthetic high-redox-potential mediators with different oxidation routes and chemical natures, while computer-aided evolution experiments were run in parallel to guide benchtop mutagenesis, without compromising protein stability. Through this strategy, the ET1 of the evolved HRPL increased from 740 to 790 mV, with a concomitant improvement in thermal and acidic pH stability. The kinetic constants for high-redox-potential mediators were markedly improved and were then successfully tested within laccase mediator systems (LMSs). Two hydrophobic substitutions surrounding the T1Cu site appeared to underlie these effects, and they were rationalized at the atomic level. Together, this study represents a proof-of-concept of the joint elevation of the ET1, redox mediator activity, and stability in an HRPL, making this versatile biocatalyst a promising candidate for future LMS applications and for the development of bioelectrochemical devices.This work was supported by the European Union project Bioenergy-FP7-PEOPLE-2013-ITN-607793, the COST Action [CM1303 Systems Biocatalysis],the Swedish Energy Agency (44707-1), the Knowledge Foundation (20170168), and the Spanish Government [BIO2016-79106-R-Lignolution and CTQ2016-79138-R].We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Increasing Redox Potential, Redox Mediator Activity, and Stability in a Fungal Laccase by Computer-Guided Mutagenesis and Directed Evolution

Fungal high-redox-potential laccases (HRPLs) are multi-copper oxidases with a relaxed substrate specificity that is highly dependent on their binding affinity and redox potential of the T1Cu site (E-T1). In this study, we combined computational design with directed evolution to tailor an HRPL variant with increased E-T1 and activity toward high-redox-potential mediators as well as enhanced stability. Laccase mutant libraries were screened in vitro using synthetic highredox-potential mediators with different oxidation routes and chemical natures, while computer-aided evolution experiments were run in parallel to guide benchtop mutagenesis, without compromising protein stability. Through this strategy, the E-T1 of the evolved HRPL increased from 740 to 790 mV, with a concomitant improvement in thermal and acidic pH stability. The kinetic constants for high-redox-potential mediators were markedly improved and were then successfully tested within laccase systems (LMSs). Two hydrophobic substitutions surrounding the T1Cu site appeared to underlie these effects, and they were rationalized at the atomic level. Together, this study represents a proof-of-concept of the joint elevation of the E-T1, redox mediator activity, and stability in an HRPL, making this versatile biocatalyst a promising candidate for future LMS applications and for the development of bioelectrochemical devices