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Published April 19, 2019 | Supplemental Material + Published
Journal Article Open

The generation of thermostable fungal laccase chimeras by SCHEMA-RASPP structure-guided recombination in vivo

Abstract

Fungal laccases are biotechnologically relevant enzymes that are capable of oxidizing a wide array of compounds, using oxygen from the air and releasing water as the only byproduct. The laccase structure is comprised of three cupredoxin domains sheltering two copper centers—the T1Cu site and the T2/T3 trinuclear Cu cluster—connected to each other through a highly conserved internal electron transfer pathway. As such, the generation of laccase chimeras with high sequence diversity from different orthologs is difficult to achieve without compromising protein functionality. Here, we have obtained a diverse family of functional chimeras showing increased thermostability from three fungal laccase orthologs with ∼70% protein sequence identity. Assisted by the high frequency of homologous DNA recombination in Saccharomyces cerevisiae, computationally selected SCHEMA-RASPP blocks were spliced and cloned in a one-pot transformation. As a result of this in vivo assembly, an enriched library of laccase chimeras was rapidly generated, with multiple recombination events simultaneously occurring between and within the SCHEMA blocks. The resulting library was screened at high temperature, identifying a collection of thermostable chimeras with considerable sequence diversity, which varied from their closest parent homologue by 46 amino acids on average. The most thermostable variant increased its half-life of thermal inactivation at 70 °C 5-fold (up to 108 min), whereas several chimeras also displayed improved stability at acidic pH. The two catalytic copper sites spanned different SCHEMA blocks, shedding light on the recognition of specific residues involved in substrate oxidation. In summary, this case-study, through comparison with previous laccase engineering studies, highlights the benefits of bringing together computationally guided recombination and in vivo shuffling as an invaluable strategy for laccase evolution, which can be translated to other enzyme systems.

Additional Information

© 2019 American Chemical Society. ACS AuthorChoice - This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. Received: December 4, 2018; Published: March 21, 2019. We truly thank Prof. Frances H. Arnold (California Institute of Technology) for her guidance and support throughout this study. This work was funded by the European Union (Bioenergy-FP7-PEOPLE-2013-ITN-607793), the CSIC (project PIE-201580E042), and the Spanish Ministry of Economy, Industry and Competitiveness (projects BIO2013-43407-R.DEWRY and BIO2016-79106-R. LIGNOLUTION). The authors declare no competing financial interest.

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August 19, 2023
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