Biotechnology Letters 21: 849853, 1999.

1999 Kluwer Academic Publishers. Printed in the Netherlands.

849

Heterologous expression of lignin peroxidase of Phanerochaete

chrysosporium in Aspergillus niger

Mohamed Sami Aifa , Sami Sayadi & Ali Gargouri

Centre de Biotechnologie de Sfax, BPK3018, Sfax, Tunisia
Author for correspondence (Fax: 216-4-275970; E-mail: Raja.Gargouri@cbs.rnrt.tn)

Received 25 May 1999; Revisions requested 24 June 1999; Revisions received 26 July 1999; Accepted 27 July 1999

Key words: Aspergillus niger, heterologous expression, lignin peroxidase, Phanerochaete chrysosporium

Abstract
The lignin peroxidase (isoenzyme H8) of Phanerochaete chrysosporium was expressed in Aspergillus niger under
the control of plant nopaline synthase (NOS) promoter and terminator. H8 mRNA was produced in this heterolo-
gous system. Western blot analysis showed that the recombinant protein was of size similar to the native H8. The
extracellular lignin peroxidase activity in these primary constructs was positive, though weak (1.12 nKat mg1 ).

Introduction rochaete chrysosporium has been successfully ob-

The ligninolytic system of Phanerochaete chrysospo-
rium is comprised of a multiplicity of enzymes with
21 isoenzymes having been detected in the extra-
cellular culture of the fungus. These represent two
types of ligninolytic peroxidases (Leisola et al. 1987):
LiP (lignin peroxidase) and MnP (manganese per-
oxidase). Lignin peroxidase oxidatively cleaves the
C-C bond of the non-phenolic propyl side chain of
aromatic lignin model compounds (Gold et al. 1984).
Lignin peroxidases also attack methoxybenzenes with
a higher redox potential than other peroxidases (Ker-
sten et al. 1990). This property, in combination with a
very broad substrate specificity, makes lignin peroxi-
dases suitable for catalyzing the degradation of diverse
recalcitrant aromatic pollutants such as polyphenols in
olive mill waste water (Sayadi & Ellouz 1995).
However, lignin peroxidase production is ham-
pered by several factors such as expression of these
enzymes under nutrient limitation and unbalanced me-
dia, and the sensitivity of this basidiomycete fungus to
high shear forces in the fermentor (Kirk et al. 1978),
besides the rapid inactivation of these enzymes even in
the absence of mycelia.
Optimization of heterologous expression has been
explored in various hosts. Heterologous expression
of a lignin peroxidase and Mn peroxidase of Phane-
tained in baculovirus (Johnson & Li 1991) and As-
pergillus oryzae (Stewart et al. 1996). The transcrip-
tion of a lignin peroxidase gene of Phlebia radiata
in Trichoderma reesei was also reported, but no pro-
tein was detected even by using a specific antibody
(Saloheimo et al. 1989). In E. coli the LiP H8 (the
major lignin peroxidase of Phanerochaete chrysospo-
rium) was expressed as inactive inclusion bodies and
the activation was obtained in vitro (Doyle & Smith
1996).
The present study addresses the production of a
recombinant lignin peroxidase (LiP H8) in a tunisian
strain of Aspergillus niger, which has the ability to
partially degrade the pollutants in olive oil waste
waters.
Materials and methods
Strains, media and culture conditions
Aspergillus niger F38, a wild-type strain isolated by
Hamdi et al. (1991) and capable of degrading pollu-
tants in olive oil waste water, was used as a recipient in
the transformation experiments. Mycelia were grown
by inoculating 108 (1 ml) conidia in 1 litre Erlenmeyer
flasks containing 200 ml LB medium (1% BactoTryp-
850
tone, 0.5% NaCl, 0.5% yeast extract, pH 6). Inocu-
lated flasks were incubated for 1824 h at 30 C with
shaking at 200 rpm. The selection of transformants
was performed on a hygromycin B minimal medium
as described by Mohr & Esser (1990).
Media and culture conditions of transformants
were done according to Cove et al. (1966) with
glycerol as a carbon source.
Escherichia coli DH5 was grown and manipu-
lated as described in Maniatis et al. (1989).
Construction of recombinant H8 plasmids
The H8 cDNA (Tien & Tu 1987) was sub-cloned
in the pMTL23 vector (Chambers et al. 1988) and
called pMTH8. After digestion by BamHI+BglII re-
striction enzymes, a fragment was released from this
plasmid of 1.3 kb representing the cDNAH8 which
was inserted between NOS (nopaline synthase) pro-
moter and terminator of pGV1503. This expression
vector was obtained from Pr. Van Montagu laboratory.
The constructed plasmid was designated as pGVH8
(7.8 kb).
Plasmid pCEL40 (Waldron et al. 1985), bearing
the hygromycin B resistance marker, was used in the
co-transformation experiments. Five g of pCEL40
with 20 g of pGVH8 was used to optimize co-
transformation.
Transformation of A. niger
Protoplasts were prepared and purified from mycelia
as described by Mohr & Esser (1990) using Novozym-
234 (Novo Industries) at 5 mg ml1 in 0.6 M KCl
and cellular debris removed by filtration through glass
wool. The regeneration frequency was from 27 to
33%. Transformation was accomplished as outlined by
Wernars et al. (1987). Transformants appeared after
3 days.
DNA and RNA extraction from transformants
Transformants were propagated in liquid medium and
mycelia were harvested by filtration through gauze
before freezing at 70 C. Cells were disrupted by
grinding 1 g frozen mycelia with 2 g alumina (sigma)
and extraction in 10 ml of TE (10 mM Tris/HCl,
pH 7.5, 1 mM EDTA, pH 8). After centrifugation
and phenol/chloroform treatment, total nucleic acids
were precipitated with 2.5 volume of cold ethanol and
1/10 volume of 3 M sodium acetate, pH 5.2. The
precipitated nucleic acids were pelleted by centrifuga-
tion, solubilized in water and either treated by RNase
or precipitated by LiCl to respectively prepare DNA
or RNA. RNA was separated in formaldehyde gel as
described in Maniatis et al. (1989).
Probe
The H8 probe is a PCR fragment of 650 bp by the fol-
lowing primers: forward: 50 -CCATGGCCTTCAAGC
AGCTC; backward: 50 -CTGCAGCTGGGCTGGCAG
GTC, according to the H8 cDNA sequence (Tien & Tu
1987).
Protein extraction and analysis
Mycelium was frozen in liquid Nitrogen, ground
and the powder suspended in 4 SDS buffer (8%
SDS, 20% -mercaptoethanol, 0.2 M Tris/HCl pH 6.8
(Moukha et al. 1993). The crude proteins were ana-
lyzed by SDS-PAGE gel electrophoresis. Extracellular
proteins were concentrated either by dehydration in
dialysis bags covered by PEG 8000, or by microfil-
tration on 10 kDa centricons (Amicon). LiP activity
(oxidation of veratryl alcohol to veratraldehyde) was
measured according to Tien & Kirk (1988). West-
ern blotting was carried according to Moukha et al.
(1993), using a polyclonal anti-H8 antibody.
Results and discussion
Plasmid construction and co-transformation results
Aspergillus niger F38 strain will grow on benzenoid
aromatics but will not degrade poly-aromatics such
as the polyphenols present in olive mill waste water.
Thus the objective was to transfer the major lignin per-
oxidase gene, H8 haemoprotein from Phanerochaete
chrysosporium to A. niger F38 which is easier to
manipulate.
The LiP H8 cDNA was inserted into the pGV1503
expression vector between NOS promoter and ter-
minator, a practical construct previously shown in
Aspergillus niger (Mohr & Esser 1990). After co-
transformation with pCEL40 vector (bearing the Hy-
gromycine resistance marker), several stable clones
of recombinant A. niger, H8 LiP and HygR positive,
were obtained as shown by Southern and PCR analy-
ses. Southern hybridization with a H8 cDNA probe
(a PCR fragment of 650 bp) showed a multiple tan-
dem integration of each construction. The digestion

Fig. 1. Southern blot analysis of recombinant Aspergillus clones.
Lanes 1, 2, 3 and 4 are pGVH8 clones digested by BglII a single
cut in the plasmid. Lane 5: HindIII DNA marker. Hybridisa-
tion was performed with a H8 radioactive probe (a 650 bp PCR
fragment) the hybridisation (left) and the gel photo (right).
of the transformants genomic DNA with a restriction
enzyme cleaving a single site in pGVH8 plasmid, re-
vealed a unique band corresponding to the size of the
linearized pGVH8 (Figure 1). An analogous result was
obtained using the HygR gene as a probe, revealing
a multiple tandem integration of pCEL40 (data not
shown).
Southern and slot-blot experiments (not shown) on
transformants genomic DNA allowed estimation of
the number of H8 tandem integration events. This
copy number is very high showing more than 10
copies in the clone number 1, Figure 1.
H8 transcription in transformants
Each transformant was cultured on rich medium sup-
plemented with glycerol as a carbon source. After
three days culture, total RNA was extracted and an-
alyzed by Northern-blotting using the H8 PCR frag-
ment as a probe. The H8 RNA was absent in the non
transformed F38 (Figure 2 lane 1) and efficiently
produced from the NOS promoter (Figure 2 lane 2).
Recombinant H8 protein detection
The NOS transformant, showing the highest H8 inte-
gration, was then analyzed by western blotting using
851
Fig. 2. Northern analysis of recombinant Aspergillus clones. Total
RNA (20 g) was loaded on a formaldehyde agarose gel (1.2%).
Lane 1: F38 as negative control. Lane 2: clone 1 of NOS trans-
formants. Radioactive hybridization was done with a H8 probe (a
650 bp PCR fragment) the hybridisation (left) and the gel photo
(right).
an antibody raised against H8 LiP. Figure 3 shows a
unique protein migrating at a level slightly lower than
the major band of Phanerochaete chrysosporium ex-
tracellular broth. The polyclonal antibody recognizes
H8 and other LiP related isoenzymes, which explains
the appearance of other weak bands in P. chrysospo-
rium profile (Figure 3 lane P), while no band is seen
in the F38 control strain (Figure 3 lane 1).
We searched the lignin peroxidase activity in ex-
tracellular proteins secreted by H8 transformants and
the F38 control strain. Since the amounts of total
proteins secreted by these strains is very low, we con-
centrated 75 times the extracellular proteins from all
glycerol cultures by microfiltration on 10 kDa cen-
tricon. The F38 control strain was LiP negative and
interestingly, NOS-transformants exhibited weak but
significant amount of LiP activity: 1.125 nKat mg1
of protein for NOS-1 and 0.75 nKat mg1 for NOS-2
transformant.
These results lead to two main conclusions: (1)
since H8 protein is a pre-pro-protein and is found in
the extracellular compartment of our transformants,
this would suggest strongly that its entry in the en-
doplasmic reticulum, followed by its cleavage by
a KEX2 like protein allowing its secretion in As-
pergillus has taken the same routing as in Phane-
rochaete chrysosporium. (2) Since H8 is also a heamo-
protein, we conclude also that heam was correctly pro-
vided to the extracellular recombinant active protein,
as in Phanerochaete chrysosporium.
852
Fig. 3. Western blot analysis of recombinant Aspergillus clones.
Western analysis was based on polyclonal antibody anti H8. Lane P:
extracellular proteins of Phanerochaete chrysosporium as the pos-
itive control. Lane 1: F38 as negative control. Lane 2: clone 1 of
NOS transformants.
How to explain the contrast between a high level
of mRNA and weak recombinant LiP activity? First,
it should be stressed that our recombinant strains
contain a single LiP isoenzyme, opposed to mul-
tiple and native LiP isoenzymes in Phanerochaete
chrysosporium. Second, many serious problems are
usually encountered whatever the heterologous sys-
tem used: RNA structure hampering the translation,
protein miss-folding, incorrect post-translational mod-
ifications and protein stability. Moreover, our recombi-
nant H8 protein probably lacks veratryl alcohol which
is known to protect it against H2 O2 in Phanerochaete
chrysosporium (Haemmerli et al. 1986).
Acknowledgements
We thank Radhouane Ellouz, principal instigator of
this project, Marc Labat, Serge Moukha and Hafedh
Belguith for their advises and discussion. Serge
Moukha is also thanked for the H8 antibody. We thank
D.E. Eveleigh for critically reading the manuscript.
This work was supported by grants of the EEC project
contract CI1-CT92-0104.
References
Chambers SP, Prior SE, Barstow DA, Minton NP (1988) The pMTL
nic-cloning vectors. I. Improved pUC polylinker regions to facil-
itate the use of sonicated DNA for nucleotide sequencing. Gene
68: 139149.
Cove DJ (1966) The induction and repression of nitrate reductase
in the fungus Aspergillus nidulans. Biochim. Biophys. Acta 113:
5156.
Doyle WA, Smith AT (1996) Expression of lignin peroxidase H8
in Escherichia coli: folding and activation of the recombinant
enzyme with Ca2+ and haem. Biochem. J. 315: 1519.
Gold MH, Kuwahara M, Chiu AA, Glenn JK (1984) Purification
and characterisation of an extra-cellular H2 O2 -requiring diaryl-
proapne oxygenase from the white rot basidiomecete Phane-
rochaete chrysosporium. Arch. Biochem. Biophys. 234: 353362.
Hamdi M, Bouhamed H, Ellouz R (1991) Optimisation of the fer-
mentation of olive mill waste waters by Aspergillus niger. Appl.
Microbiol. Biotechnol. 36: 285288.
Haemmerli SD, Leisola MS, Sanglard D, Fiechter A (1986) Oxi-
dation of benzo(a) pyrene by extracellular ligninases of Phane-
rochaete chrysosporium. J. Biol. Chem. 261: 69006903.
Johnson TM, Li JK (1991) Heterologous expression and charac-
terisation of an active lignin peroxidase from Phanerochaete
chrysosporium using recombinant baculovirus. Arch. Biochem.
Biophys. 291: 371378.
Kersten PJ, Kalyanaraman B, Hammel KE, Reinhammar B, Kirk
TK (1990) Comparison of lignin peroxidase, horseradish peroxi-
dase and laccase in the oxidation of methoxybenzenes. Biochem.
J. 268: 475480
Kirk TK, Schultz E, Connors WJ, Lorenz LF, Zeikus JG (1978)
Influence of culture parameters on lignin metabolism by Phane-
rochaete chrysosporium. Arch. Microbiol. 117: 277285.
Leisola MS, Kozulic B, Meussdoerffer F, Fiechter A (1987) Ho-
mology among multiple extra-cellular peroxidases from Phane-
rochaete chrysosporium. J. Biol. Chem. 262: 419424.
Maniatis T, Fritsch EF, Sambrook J (1989) Molecular Cloning:
A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory Press.
Mohr G, Esser K (1990) Improved transformation frequency and
heterologous promoter recognition in Aspergillus niger. Appl.
Microbiol. Biotechnol. 34: 6367.
Moukha SM, Wosten HA, Mylius EJ, Asther M, Wessels JG (1993)
Spatial and temporal accumulation of mRNAs encoding two
common lignin peroxidases in Phanerochaete chrysosporium. J.
Bacteriol. 175: 36723678.
Saloheimo M, Barajas V, Niku-Paavola ML, Knowles JK (1989)
A lignin peroxidase encoding cDNA from the white-rot fungus
Phlebia radiata: characterisation and expression in Trichoderma
reesei. Gene 85: 343351.
Sayadi S, Ellouz R (1995) Roles of lignin peroxidase and man-
ganese peroxidase from Phanerochaete chrysosporium in the
decolarization of olive mill wastewaters. Appl. Env. Microbiol.
61: 10981103.
Stewart P, Whitwam RE, Kersten PJ, Cullen D, Tien M (1996) Ef-
ficient expression of Phanerochaete chrysosporium manganese
peroxidase gene in Aspergillus oryzae. Appl. Env. Microbiol. 62:
860864.
Tien M, Tu CPD (1987) Cloning and sequencing of a cDNA for a
ligninase from Phanerochaete chrysosporium. Nature 326: 520
523.
Tien M, Kirk TK (1988) Lignin peroxidase of Phanerochaete
chrysosporium. Meth. Enzymol. 161: 238249.
 853

Waldron C, Murphy EB, Roberts JL, Gustafson GD, Armour SL, Wernars K, Goosen T, Swart K, Hondel CAMJJ van den, Broek
Malcom SK (1985) Resistance to hygromycin B: a new marker HWJ van den (1987) Cotransformation of Aspergillus nidulans:
for plant transformation studies. Plant Mol. Biol. 5: 103108. a tool for replacing fungal genes. Mol. Gen. Genet. 209: 7177.