Review of forests, wood products and wood biotechnology ... - GWDG

313
P. ostreatus produces in solid state fermentation as well as in submerged
fermentation both types of enzymes, laccases and peroxidases, at reasonable yields
(Rühl et al. 2007, 2008). In contrast, C. cinerea can naturally produce laccase but
this is usually at quite low levels even if an inducer such as copper is added
(Fig. 11). In industry, strains of the white-rot Trametes versicolor are used for laccase
production but there is also an interest in recombinant production through gene
cloning and protein expression in heterologous hosts. Gene cloning and
heterologous protein expression gain special attention when the protein of interest
comes from a fungus of which no fermentation system has been established
(Kilaru 2006, Rühl et al. 2007) and there are many different fungi possessing very
interesting laccases (Hoegger et al. 2006). Attempts in the past of recombinant
laccase production made use of ascomycetes fungi such as the baker´s yeast
Saccharomyces cerevisiae or the filamentous fungus Aspergillus nidulans. However,
laccases from Agaricomycetes tend to become modified in the heterologous hosts
by attaching in an inappropriate way sugar-residues to the proteins. Such wrong
glycosylation unfortunately affects negatively the yields, stability and enzymatic
characteristics of the laccases (Kilaru 2006, Rühl et al. 2007).
Laccase acivity (mU/ml)
100
80
60
40
20
Addition
of Cu
1 3 5 6 7 8 9 10 11 12 13 15
Days of incubation
Control
Cuinduction
Figure 11: Laccase
production by a
strain of
Coprinopsis
cinerea in standing
submerged culture
with (red line) and
without (blue line)
addition of copper
to the cultures
(data are from Rühl
et al. 2007).
To overcome the problem given by recombinant expression in ascomycetes, it was
apparent to test heterologous expression within species of the Agaricomycetes
(Kilaru 2006, Rühl et al. 2007). We chose the easy to transform C. cinerea and
developed a system of efficient expression of laccase genes under control of a
constitutive highly active promoter from the edible species Agaricus bisporus (gpdII