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1.7 Conclusions and Outlook 17

environments. However, this situation has changed with the development of
directed evolution. In the last 10 years, a plethora of ideas for the construction of
ligninolytic mutant libraries in S. cerevisiae has arisen to improve secretion, activity,
and thermostability to broaden pH activity profiles and even to induce activity in
blood or in organic co-solvents. And this is just the beginning. However, many
challenges remain despite the great potential of these evolutionary platforms.
We will have to deal with serious bottlenecks, such as the poor production of
the mutants in different heterologous hosts (practical alternatives may exploit
tandem expression systems with S. cerevisiae as a host for directed evolution and
P. pastoris or Aspergillus niger for overexpression of the evolved variants [84]); the
delicate balance between activity and stability when introducing beneficial (but
destabilizing) mutations; and the difficulty of maintaining the redox potential
of mutants after several rounds of evolution. In this context, the use of neutral
genetic drift for the creation of polymorphic mutant libraries will open new
adaptive pathways to enhance the stability or even the catalytic promiscuity of these
oxidoreductases. Finally, the combination of directed evolution with both hybrid
and rational approaches, including computational studies, will permit to convert
this versatile array of high-redox potential oxidoreductases into real biotechnological
products.

Acknowledgments

This chapter is based on research funded by projects: FP7-3D-Nanobiodevices,
NMP4-SL-2009-229255; FP7-KBBE-2010-4-26537, Peroxicats; FP7-KBBE-2013-7-
613549, Indox; COST Actions CM0701 CM0701-Cascat and CM1303-Sysbiocat;
and a National Project BIO2010-19697-Evofacel).

Abbreviations

AAD aryl-alcohol dehydrogenase
AAO aryl-alcohol oxidase
′
ABTS 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
CDH cellobiose dehydrogenase
CIP Coprinopsis cinerea peroxidase
CLERY combinatorial libraries enhanced by recombination in yeast
DMP 2,6-dimethoxyphenol
EDTA ethylenediamine-tetra-acetic acid
GLX glyoxal oxidase
HRP horseradish peroxidase
HRPLs high-redox potential laccases
HTS high-throughput screening
I Concentration of an inhibitory substance at which the enzyme
50
retains 50% of its initial activity