Abstract

SummaryAlthough the functional expression of fungal laccases in Saccharomyces cerevisiae has proven to be complicated, the replacement of signal peptides appears to be a suitable approach to enhance secretion in directed evolution experiments. In this study, twelve constructs were prepared by fusing native and evolved α‐factor prepro‐leaders from S. cerevisiae to four different laccases with low‐, medium‐ and high‐redox potential (PM1L from basidiomycete PM1; PcL from Pycnoporus cinnabarinus; TspC30L from Trametes sp. strain C30; and MtL from Myceliophthora thermophila). Microcultures of the prepro‐leader:laccase fusions were grown in selective expression medium that used galactose as both the sole carbon source and as the inducer of expression so that the secretion and activity were assessed with low‐ and high‐redox potential mediators in a high‐throughput screening context. With total activity improvements as high as sevenfold over those obtained with the native α‐factor prepro‐leader, the evolved prepro‐leader from PcL (αPcL) most strongly enhanced secretion of the high‐ and medium‐redox potential laccases PcL, PM1L and TspC30L in the microtiter format with an expression pattern driven by prepro‐leaders in the order αPcL > αPM 1L ~ αnative. By contrast, the pattern of the low‐redox potential MtL was αnative > αPcL > αPM 1L. When produced in flask with rich medium, the evolved prepro‐leaders outperformed the αnative signal peptide irrespective of the laccase attached, enhancing secretion over 50‐fold. Together, these results highlight the importance of using evolved α‐factor prepro‐leaders for functional expression of fungal laccases in directed evolution campaigns.

Highlights

  • Fungal laccases (EC 1.10.3.2, benzenediol:oxygen oxidoreductases) catalyse the oxidation of phenols, aromatic amines and other compounds, with the concomitant reduction of molecular oxygen to water (Solomon et al, 1996; Gianfreda et al, 1999; Alcalde, 2007)

  • Given this broad substrate range and their minimal requirements, fungal laccases belong to the elite of oxidases that can be employed in very distinct areas of biotechnology, from organic synthesis to novel green processes and beyond (Riva, 2006; Kunamneni et al, 2008a,b; Mate and Alcalde, 2017)

  • The canonical pre-leader is implicated in the translocation of the nascent secretory protein, which is removed from the endoplasmic reticulum (ER) membrane by the action of a signal peptidase between residues 19 and 20

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Summary

Summary

The functional expression of fungal laccases in Saccharomyces cerevisiae has proven to be complicated, the replacement of signal peptides appears to be a suitable approach to enhance secretion in directed evolution experiments. With total activity improvements as high as sevenfold over those obtained with the native a-factor prepro-leader, the evolved prepro-leader from PcL (aPcL) most strongly enhanced secretion of the high- and medium-redox potential laccases PcL, PM1L and TspC30L in the microtiter format with an expression pattern driven by prepro-leaders in the order aPcL > aPM1L ~ anative. When produced in flask with rich medium, the evolved prepro-leaders outperformed the anative signal peptide irrespective of the laccase attached, enhancing secretion over 50-fold. Together, these results highlight the importance of using evolved a-factor prepro-leaders for functional expression of fungal laccases in directed evolution campaigns

Introduction
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