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8 1 Directed Evolution of Ligninolytic Oxidoreductases

and enzymatic co-factor regeneration. Recently, the MtL-R2 mutant was converted
into an alkalophilic fungal laccase [43]. Accordingly, a high-throughput screening
(HTS) assay based on the activity ratio at pH 8.0 to 5.0 was used as the main
discriminatory factor. Screening the laccase mutant libraries at alkaline pH while
conserving activity at acidic pHs led to a shift in the pH activity profiles that was
accompanied by improved catalytic efficiency at both pH values (31-fold at pH 7.0
and 12-fold at pH 4.0). The final variant obtained in this evolution experiment (the
IG-88 mutant) retained 90% of its activity at pH 4.0–6.0 and 50% at pH 7.0, and
some activity was even detected at pH 8.0.
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.

1.4.3
Directed Evolution of Ligninolytic High-Redox Potential Laccases (HRPLs)

Two HRPLs from the basidiomycete PM1 laccase (PM1L) and Pycnoporus cinnabar-
inus laccase (PcL) were subjected to parallel comprehensive directed evolution
in order to achieve functional expression in S. cerevisiae while conserving their
thermostability [44]. PM1L was tailored during eight cycles of directed evolution
combined with semirational/hybrid approaches [45]. The native laccase signal
sequence was replaced by the α-factor prepro-leader from S. cerevisiae and it was
evolved in conjunction with the mature protein, adjusting both elements for a
successful exportation by yeast. After screening over 50 000 clones, this approach
led to the generation of a highly active, soluble, and thermostable HRPL. The total
improvement in activity achieved was as high as 34 000-fold relative to the parental
type, an effect brought about by the synergies established between the evolved
prepro-leader and the mature laccase. Several strategies were employed to main-
tain the stability of the laccase while enhancing its activity and secretion during
evolution: (i) screening for stabilizing mutations [46]; (ii) mutational exchange with
beneficial PcL mutations; and (iii) mutational recovery of beneficial mutations with
a low likelihood of recombination [44, 47].
The final mutant generated in this process (the OB-1 variant with 15 mutations
accumulated both in the prepro-leader and in the mature protein) exhibited
−1
secretion levels of ∼8mgl , and it was very active and stable over a range of
◦
temperatures (T 50 ∼ 73 C) and pH values, as well as in the presence of organic
co-solvents [45]. OB-1 was recently subjected to four further rounds of directed
evolution and saturation mutagenesis in order to achieve activity in human blood,
a milestone that will allow it to be used in a wide array of exciting biomedical and
bioanalytical applications [48]. The inherent inhibition of laccase by the combined
action of high NaCl concentrations (∼140 mM) and the alkaline pH (∼7.4) of blood
was overcome by using an ad hoc HTS assay in a buffer that simulated blood
but lacked coagulating agents and red blood cells. Bearing in mind that HRPLs
are not active at neutral pH, the selective pressure was enhanced in successive
rounds of evolution, starting at pH 6.5 and finishing at physiological pH. The final