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1.6 Saccharomyces cerevisiae Biomolecular Tool Box 15

interest. To face this challenge, we have developed a new domain-random
mutagenesis/recombination method based on S. cerevisiae physiology known
as MORPHING (Mutagenic Organized Recombination Process by Homologous
IN vivo Grouping) [73]. This method has also been useful to evolve the signal
peptide of UPOs for functional expression (Figure 1.5). MORPHING is highly
suited to the exploration of limited targeted regions (even those <30 residues
long), which has allowed us to reveal certain structural determinants involved
in H O inhibition. The combination of MORPHING with classical DNA-
2 2
diversity evolution methods (e.g., random mutagenesis, DNA recombination,
saturation mutagenesis), as well as with the mutational recovery of certain
positions by site-directed mutagenesis, has enabled us to obtain final VP
variants with longer half-lives after several rounds of evolution and screening
(Gonzalez-Perez, D., et al., unpublished material).
Our laboratory is also studying the directed evolution of a new type of potentially
ligninolytic peroxidase classified as unspecific peroxygenase, UPO (EC 1.11.2.1).
UPO was initially defined as a heme-thiolate peroxidase, exhibiting both per-
oxidative and peroxygenative activities toward aromatic compounds (aromatic
peroxygenase, also referred to as APO [26, 75]). However, more recent studies have
described the monooxygenase activity of UPO toward aliphatic compounds (UPO,
[76, 77]). The peroxygenative (oxygen-transfer) activity is of particular significance
as UPO can behave as a self-sufficient monooxygenase performing regio- and
enantio-specific oxyfunctionalizations that are of great interest for organic synthe-
ses. UPO, like other ligninolytic oxidoreductases described in this chapter, was not
functionally expressed in heterologous hosts for directed evolution. Very recently,
this problem was tackled by subjecting it to five cycles of evolution in S. cerevisiae,
which enhanced its secretion up to 6500 ABTS units l −1 (8 mg/L) with the help
of a dual colorimetric HTS assay to protect the synthetic abilities of the enzyme
(i.e., both peroxidative and peroxygenative activities were simultaneously screened,
[53]). In the same study, MORPHING was applied to independently evolve the
native signal sequence, which was finally attached to the native mature UPO to be
able to consistently establish the secretion and catalytic efficiencies by comparing
the native and evolved UPO secreted by yeast [53, 73].

1.6
Saccharomyces cerevisiae Biomolecular Tool Box

Most of the directed evolution studies described in this chapter have been carried
out in S. cerevisiae because it is not only important for the functional expression
of evolved mutants but also is fundamental to create methods to generate
DNA diversity, which introduces and recombines new point mutations [78].
In addition to providing an efficient secretory machinery that permits complex
post-translational processing, S. cerevisiae offers a high frequency of homologous
DNA recombination. This feature is extremely useful to devise in vivo diversity
strategies, from DNA shuffling to the assembly of cassette mutant genes for