Page 34 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

10 1 Directed Evolution of Ligninolytic Oxidoreductases

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halides and decreases in T values (4.8 and 3.6 C for both mutations, respectively).
50
When a crossroad is reached between activity and stability, it is difficult to further
evolve the protein as it does not tolerate the introduction of new sets of beneficial
but destabilizing mutations without compromising its structure and function. We
are currently attempting to improve the stability of this variant by introducing new
stabilizing mutations, such as A361T and S482L from the 16B10 mutant of PM1L
[46]. These results reflect the subtle equilibrium between activity and stability when
evolving enzymes in the laboratory for nonnatural functions, consistent with the
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observations in earlier directed evolution studies. For example, a decrease by 10 C
in the T 50 was obtained following the directed evolution of P450 BM-3 from Bacillus
megaterium to convert it into an alkane monooxygenase [49–51].
To conclude this mutational pathway, PM1L 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 PM1L to such inclement conditions
affected only 0.4% of the amino acid sequence.
The evolution of the HRPL from PcL was tackled using a similar approach to
that described for PM1L (i.e., joint evolution of the α-factor prepro-leader and
the mature protein). Six cycles of directed evolution were performed to obtain an
enzyme that could be readily expressed by yeast (with secretion levels of ∼2mgl −1
[52]). A multiple HTS assay based on the oxidation of natural and synthetic
redox mediators was employed to discriminate between mutants with improved
activities against phenolic and nonphenolic compounds. The final variant of this
process (the 3PO mutant, containing 14 mutations) retained its thermostability
while significantly broadening its pH activity profile. Notably, the breakdown in
secretion and activity was accomplished by fusing the evolved prepro-leader to
the native PcL. The evolved signal sequence improved secretion 40-fold, while the
mutations that accumulated in the evolved mature protein were responsible for
a ∼14-fold enhancement in the k , together with an improved secretion/folding
cat
of theenzyme(∼14-fold improvement). The directed evolution of signal peptides
to enhance secretion and their additional attachment to nonevolved proteins is a
valuable strategy for the directed evolution of other ligninolytic oxidoreductases
(unspecific peroxygenases, see below [53]).
The sequence identity between PcL and PM1L is over 77%, which facilitated
mutational exchange between the two parallel evolution pathways and allowed us
to switch protein sequence blocks to create chimeric proteins of HRPLs with hybrid
or even enhanced features. To favor multiple crossover events between laccase
scaffolds, in vitro and in vivo DNA recombination methods were combined in a
single evolutionary step (see Section 1.6). Chimeras with up to six crossover events
per sequence were identified, which generated active laccase hybrids with combined
characteristics in terms of substrate affinity, pH activity, and thermostability [54].
Interestingly, some chimeras showed higher thermostabilities than the original