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of the corresponding gene (
Conesa et al., 2002
). Other studies focused on
the MnP of C. subvermispora or the VP of P. eryngii in A. nidulans, which
demonstrates the possibility of using a fungal system to heterologously
produce MnPs or VP, in spite of a lower productivity (
Eibes et al., 2009;
Larrondo et al., 2001
). To date, the overproduction of LiPs in fungal hosts
has not yet been achieved and remains a challenge, despite some earlier work
(
Conesa et al., 2000
).
A few studies have been conducted to produce CDH (belonging to the
FOLy family LO3) in various yeast systems, but no results are available to
examine the efficiency of filamentous fungi to produce CDH. To analyse the
possibility of homologously producing CDH, the P. cinnabarinus system
has been tested in our lab (unpublished data). The corresponding gene was
previously cloned from a genome library (
Dumonceaux et al., 1998
)and
fused under the control of the gpdAgenepromoterofS. commune. The first
results in flask cultures revealed that CDH was efficiently produced and
yielded approximately 10 mg/L. The heterologous expression of the same
gene in P. pastoris yielded a functional CDH in high yield and the optimi-
zation of this expression in a bioreactor increased the production up to
350 mg/L (Bey et al., unpublished results).
In contrast to enzymes acting directly on lignin (LO family), only sparse
studies are available on the production of LDA enzymes in filamentous
fungi. For example, the extracellular AAO LDA1 form P. eryngii was cloned
in A. nidulans under the control of the homologous alcohol dehydrogenase
promoter (
Varela et al., 2001
). This production resulted in 10-50 times higher
levels of activity compared with the wild-type P. eryngii cultures while
the biochemical properties were conserved for the recombinant enzyme.
A. nidulans was also used to heterologously produce the Glox (LDA3) of
the basiodiomycete fungus P. chrysosporium, using the glucoamylase gene
promoter. Production yields of 10-20 mg/L were 50 times greater than that
found in optimized P. chrysosporium cultures (
Kersten et al., 1995
). To
identify the catalytic residues of P. chrysosporium Glox by targeted mutagen-
esis, mutants have been heterologously expressed in P. pastoris, which pro-
duced as much as 2 g/L of protein under conditions of high-density
methanol-induced fermentation (
Whittaker et al., 1999
). Finally, the produc-
tion of the A. nidulans glucose oxidase (LDA6) was improved by homologous
overproduction using the promoter of the acidic xylanase-encoding gene
xlnB(
Luque et
al., 2004
). Concerning this last family, a wide diversity of
oxidases from ascomycetes and basidiomycetes fungi were expressed success-
fully in P. pastoris. For example, using a codon optimization approach, the
glucose oxidase from Penicillium notatum was recently expressed at up to
2.5 g/L (
Gao et al., 2012
).
