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16 1 Directed Evolution of Ligninolytic Oxidoreductases

metabolic engineering and synthetic biology studies (some of the DNA diversity
methods based on S. cerevisiae physiology for library creation are summarized
in Figure 1.5). In all cases, the methodology involves engineering overlapping
regions that favor in vivo splicing and recombination in yeast. Some of the most
versatile methods are outlined as follows:
(i) In vivo overlap extension (IVOE) is a fast and simple protocol to perform site-
directed mutagenesis, combinatorial (saturation) mutagenesis, and insertion
and deletion mutagenesis [79, 80] (Figure 1.5a). It has been used to construct
combinatorial saturation mutagenesis libraries of MtL in order to explore
the functionality of the C-terminal plug [81]; to engineer truncated variants
of VP and PM1L to analyze the processing of the α-factor prepro-leader
[72, 74]; to replace native signal peptides in PcL and UPO ([52, 53]; to perform
site-directed mutagenesis and saturation mutagenesis of MtL for evolution
toward organic co-solvent tolerance and activity at an alkaline pH [40, 43]; and
during directed PM1L evolution for secretion in yeast and activity in blood
[45, 48].
(ii) In vivo assembly of mutant libraries (IvAM) combines the mutational profiles
of different polymerases in a single round of evolution [42] (Figure 1.5b). It
has been used comprehensively during the evolution of MtL, PM1L, UPO,
and VP [40, 43, 45, 48, 53, 72].
(iii) StEP (staggered extension process) + in vivo DNA shuffling was useful
to enhance the thermostability of VP without compromising activity [46]
(Figure 1.5c).
(iv) In vitro DNA shuffling + in vivo DNA shuffling (also known as CLERY
(combinatorial libraries enhanced by recombination in yeast) [82]) has been
used to generate chimeric laccases with large sequence divergence [54]
(Figure 1.5d). Both (iii) and (iv) are combinations of in vivo and in vitro
recombination methods for library creation.
(v) MORPHING is a focused directed evolution method used to introduce
random mutations and recombination of specific regions (Figure 1.5e). It
was highly suited to the directed evolution of VP for oxidative stability and
for the directed evolution of the signal peptide of UPO for its secretion in
yeast [73].
(vi) DNA assembler allows the construction of complex metabolic pathways by
assembling whole genes and promoters with common overlapping regions
[83] (Figure 1.5f). This approach is currently being used in our laboratory
to construct synthetic ligninolytic pathways in yeast (Gonzalez-Perez D. and
Alcalde M., unpublished material).

1.7
Conclusions and Outlook
Some years ago, the engineering of the ligninolytic consortium of oxidoreductases
was too littered with hurdles to apply these enzymes to aggressive industrial