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2 1 Directed Evolution of Ligninolytic Oxidoreductases

from its natural environment and introduced into a specific biotechnological
location (e.g., the transformation of a hydrophobic compound in the presence of
co-solvents or at high temperatures), its molecular structure may not tolerate the
extreme operational conditions and may unfold becoming inactive. Unfortunately,
the enzymes that cells use to regulate strict metabolic pathways and that promote
fitness and survival in nature are not always applicable to the harsh requirements
of many industrial processes.
The development of the polymerase chain reaction (PCR) in the early 1980s
heralded a biotechnological revolution for protein engineers, allowing us for
the first time to manipulate and design enzymes by site-directed mutagenesis
supported by known protein structures: the so-called rational design. However,
further advances were frustrated owing to the limited understanding of protein
function and the lack of protein structures available at the time. Nevertheless, the
following decade saw a second biotechnological revolution with the development
of directed molecular evolution. This powerful protein engineering tool does
not require prior knowledge of protein structure to enhance the known features
or to generate novel enzymatic functions, which are not generally required in
natural environments. The key events of natural evolution (random mutation,
DNA recombination, and selection) are recreated in the laboratory, permitting
DNA diversity
Random Mutation
mutagenesis
Linearized
plasmid
New
generation Parental genes
Best mutant hit Recombination
(parent for next generation)
Cloning and transformation
in heterologous host

Screening assay

Clone growth
Functional expression
in HT format
Figure 1.1 Directed molecular evolution. (typically S. cerevisiae or E. coli); (ii) a reli-
The basic premises to carry out a success- able high-throughput (HT)-screening assay;
ful directed evolution experiment are (i) and (iii) the use of different molecular tools
a robust heterologous expression system for the generation of DNA diversity.