Biology Reference
In-Depth Information
IV. RECOMBINANT PRODUCTION OF LIGNINASES
IN FUNGAL HOSTS
Saprophytic filamentous fungi are an important source of enzymes secreted
in the extracellular compartment to degrade wood in natural conditions. As
some of them secrete very small amounts of ligninases, efficient host system
tools are needed to produce larger amount of proteins for further biochemi-
cal and structural characterization and/or exploitation in biotechnological
applications. In addition, genetic expression systems are necessary to modify
enzymes to meet the physicochemical properties needed to improve industrial
processes and/or to study the exact function of enzymes in the delignification
process. Filamentous fungi, which are multicellular eukaryotic organisms, are
suitable hosts for several reasons. They were first used as alternative systems of
expression to bacteria, because of post-translational maturation steps that
could not be performed in Escherichia coli. Compared with mammalian sys-
tems, fungi need less time to attain the various stages corresponding to gene
expression and protein production. Yeast (Pichia pastoris and Yarrowia lipo-
lytica) and filamentous fungi (including Aspergilli, for example A. awamori,
A. niger, A. oryzae and Trichoderma reesei) are widely used for their ability to
secrete large quantities of proteins. Fungal host strains have been engineered
to decrease their proteolytic activities. In the same way, strains deficient for
certain abundantly secreted homologous proteins have been obtained to alle-
viate the secretory pathway, for example glucoamylase A for Aspergilli and
cellobiohydrolase I for Trichoderma.
To improve the production yield of ligninases and possibly use them for
industrial applications, filamentous fungi have been shown to be very attrac-
tive systems. They have a natural ability to produce large amounts of
proteins, such as T. reesei, which can produce up to 50 g/L of cellulases
(
Eveleigh and Montenecourt, 1979
)orAspergillus species such as A. niger
and A. oryzae, which are able to produce several specific proteins at up to
30 g/L for commercial utilization (
Punt et al., 2002
).
Laccases (LO1 family) have been extensively studied for heterologous
production in filamentous fungi. Although production of several laccases
from basidiomycetes has been reported in yeast expression systems, yields
were rather low, for example, up to 12 U/mL for the T. versicolor laccase in
the yeast Pichia methanolica (
Guo et al., 2006
). The Aspergillus expression
system was more efficient, yielding from some 10 mg/L to more than
100 mg/L of laccase in the extracellular medium (for an exhaustive list of
heterologously produced laccases; see
Piscitelli et al., 2010
). The production
of the P. cinnabarinus laccase has been achieved in several hosts, including
yeast and ascomycete fungi, to compare the protein production performance
