Directed Evolution of Medium-Redox Potential Laccases

Directed Evolution of Medium-Redox Potential Laccases 

The first successful example of the directed evolution of fungal laccase involved the laccase from the thermophile ascomycete Myceliophihora thermophila laccase (MtL). This study led to subsequent directed evolution experiments in S. cerevisiae with several high-redox potential ligninolytic oxidoreductases (see below). MtL was subjected to 10 cycles of directed evolution to enhance its functional expression in S. cerevisiae [38]. The best performing variant of this process (the T2 mutant that harbored 14 mutations) exhibited a total improvement of 170-fold in activity its expression levels were enhanced 8-fold and the around 22-fold. The 

After 20 generations, the successful in vitro evolution of MtL can be attributed to the plasticity and robustness of this thermostable protein, highlighting that there may be an additional margin for further engineering. 

Directed Evolution of Ligninoiytic High-Redox Potential Laccases (HRPLs) 

To conclude this mutational pathway, PMIL was sculpted by 12 rounds of directed evolution, in which it accumulated 22 mutations (8 silent) throughout the entire fusion gene. Beneficial mutations that enhanced secretion or activity were located in the signal prepro-leader (5 mutations) and the mature protein (7 mutations), respectively. Significantly, only two mutations located in the second coordination sphere of the T1 copper site conferred tolerance to blood. Therefore, the re-specialization required to adapt the PMIL to such inclement conditions affected only 0.4% of the amino acid sequence. 

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