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MINI-REVIEW

Fungal microsomes in a biotransformation perspective:


protein nature of membrane-associated reactions
Kateina Svobodov & Hana Mikeskov &
Denisa Petrkov
Received: 24 July 2013 / Revised: 16 October 2013 / Accepted: 17 October 2013 / Published online: 5 November 2013
#Springer-Verlag Berlin Heidelberg 2013
Abstract Microsomal fraction of fungal cells grabs the atten-
tion of many researchers for it contains enzymes that play a
role in biotechnologically relevant processes. Microsomal en-
zymes, namely, CYP450s, were shown to metabolize a wide
range of xenobiotic compounds, including PAHs, PCBs, di-
oxins, and endocrine disruptors, and take part in other fungal
biotransformation reactions. However, little is known about the
nature and regulation of these membrane-associated reactions.
Advanced proteomic and post-genomic techniques make it
possible to identify larger numbers of microsomal proteins
and thus add to a deeper study of fungal intracellular processes.
In this work, proteins that were identified through a shotgun
proteomic approach in fungal microsomes under various cul-
ture conditions are reviewed. However, further research is still
needed to fully understand the role of microsomes in fungal
biodegradation and biotransformation reactions.
Keywords Fungal microsomes
.
Cytochrome P450
.
Biodegradation
.
Microsomal proteins
.
Proteomics
Introduction
The biodegradation potential of white rot and other filamen-
tous fungi has been extensively studied during the last
decades. The studies were focused mainly on fungal extracel-
lular oxidative enzymes, their ability to oxidize various per-
sistent organic compounds, and the elucidation of degradation
pathways. Based on the results of inhibitor studies and me-
tabolite identification, however, involvement of fungal cyto-
chrome P450 (CYP450) system and other intracellular en-
zymes in the biodegradation of organopollutants was sug-
gested (Mougin et al. 1996; Mougin et al. 1997; Covino
et al. 2010; Prieto et al. 2011; vanarov et al. 2012;
Kesinov et al. 2012). Filamentous fungi have been then
shown to possess a high diversity of CYP450 systems with a
broad substrate activity (Vatsyayan et al. 2008; Lah et al.
2008). Their relation to fungal metabolism of xenobiotic
chemicals has been reviewed previously (renar and Petri
2011; Peng et al. 2008). It underlined the need of better
understanding of fungal intracellular processes and opened a
new line for fungal biodegradation research.
In this work, findings supporting degradation ability and
biotransformation potential of fungal microsomal enzymes are
summarized. Fungal microsomes are equivalent to subcellular
membrane fractions that are obtained from homogenized fun-
gal mycelium by differential centrifugation as described by
Cinti et al. (1972) for rat microsomes or through ultracentri-
fugation steps (Machida and Saito 1993; Mougin et al. 1997).
The purity of the microsomal preparations can be checked by
enzymatic marker assays as described in Jauregui et al.
(2003). Endoplasmic reticulum marker NADH-cytochrome c
reductase has been determined as a marker activity for micro-
somal fractions.
To clarify the nature of microsomal processes, two types of
studies have been nowadays carried out in fungi: narrowly
focused works on functions of individual enzymes and
proteome-wide studies. For example, mechanisms involved
in the recognition of aromatic compounds by the fungal
CYP450 were studied in the model fungus Phanerochaete
chrysosporium (Syed et al. 2011). Contrary to that, a large
number of microsomal proteins were identified in Aspergillus
niger (DeOliveira et al. 2010; DeOliveira et al. 2011) using
K. Svobodov (*)
:
H. Mikeskov
Laboratory of Environmental Biotechnology, Institute of
Microbiology ASCR, v.v.i., Videnska 1083,
14220 Prague, Czech Republic
e-mail: ksvobod@biomed.cas.cz
H. Mikeskov
Institute of Chemical Technology Prague,
Faculty of Food and Biochemical Technology, Technick 5,
160 28 Prague 6, Czech Republic
D. Petrkov
Laboratory of Cell Signalization, Institute of Microbiology ASCR,
v.v.i., Videnska 1083, 14220 Prague, Czech Republic
Appl Microbiol Biotechnol (2013) 97:1026310273
DOI 10.1007/s00253-013-5347-2
advanced proteomic techniques. Both approaches could help
to enhance the understanding of fungal intracellular processes.
In this work, changes in membrane enzymes during biodeg-
radation reactions and identification of microsomal proteins
are discussed to give an insight into the nature and regulation
of microsomal biodegradation and biotransformation process-
es. Advanced mapping of microsomal proteomes by high-
throughput proteomics is especially highlighted as a useful
tool for microsomal protein analyses.
Biodegradation potential of fungal microsomes
The implication of microsomal enzymes in xenobiotic detox-
ification and degradation is summarized in this chapter. A
short review is given in Table 1.
Several biodegradation studies suggested the involvement
of intracellular enzymes in the biodegradation reactions. The
work of Masaphy et al. (1996a) indicated that CYP450-
mediated benzo[a]pyrene hydroxylase activity in both micro-
somal and soluble fractions of the white rot fungus P.
chrysosporium could play a role in the xenobiotic transfor-
mation by this fungus. The biotransformation of an insecticide
lindane and herbicide atrazine by the liquid cultures of
P. chrysosporium has been drastically reduced by
1-aminobenzotriazole (a CYP450 inactivator) (Mougin et al.
1996; Mougin et al. 1997). Conversely, phenobarbital (a P450
inducer) did not significantly increase lindane breakdown.
Various inhibition studies also affirmed the implication of
P. chrysosporium CYP450s (PcCYPs) in the degradation of
nonylphenol (Subramanian and Yadav 2009) and pentachlo-
rophenol (PCP) (Ning and Wang 2012), hydroxylation of
xenobiotics (Hiratsuka et al. 2005; Teramoto et al. 2004a,b),
and oxidation of the chlorinated pesticide endosulfan
(Kullman and Matsumura 1996). To study substrate specific-
ity of individual PcCYPs, 120 yeast clones expressing indi-
vidual CYP450s were screened for transformation of dioxins
(Kasai et al. 2010). Out of 40 positive clones, a microsomal
PcCYP designated as PcCYP11a3 showed the highest activ-
ity. It catalyses the hydroxylation of 2,3- dichlorodibenzo-p-
dioxin and has the highest activity towards polychlorinated
dioxins among the known CYP450s derived from microor-
ganisms. Recently, a genome-wide gene induction strategy
revealed multiple PcCYPs responsive to individual classes
of xenobiotics (Syed et al. 2010). CYP5136A3 then showed
a common responsiveness and catalytic versatility towards
endocrine-disrupting alkylphenols and polycyclic aromatic
hydrocarbons (PAHs; Syed et al. 2011). Metabolic pathways
of PAHs by fungal P450 monooxygenases were already
reviewed in the work of Peng et al. (2008).
Next to P. chrysosporium, the potential of microsomal
fractions to metabolize xenobiotics was studied in other fungi,
too. Cytosolic and microsomal fractions of Cunninghamella
elegans were assayed for activities of cytochrome P450
monooxygenase, aryl sulfotransferase, glutathione S-transfer-
ase, UDP-glucuronosyltransferase, and N-acetyltansferase
and connected with the physiological versatility of the fungus
in the metabolism of xenobiotics (Zhang et al. 1996). Eilers
et al. (1999) showed that the metabolism of 2,4,6-trinitrotol-
uene (TNT) in Bjerkandera adusta may include CYP450-
dependent reactions.
Bezalel et al. (1997) examined the enzymatic mechanisms
involved in the degradation of phenanthrene by Pleurotus
ostreatus. CYP450 activity was detected in both cytosolic
and microsomal fractions of the fungus; however, it was
inhibited differently by the CYP450 inhibitors 1-
aminobenzotriazole, SKF-525A(proadifen), and carbon mon-
oxide. The experiments indicated the involvement of cyto-
chrome P450 monooxygenase and epoxide hydrolase in the
initial oxidation of phenanthrene to form phenanthrene
trans-9,10-dihydrodiol.
The extracellular and microsomal fractions of P. ostreatus
7989 were tested for in vitro degradation of five pesticides
(Jauregui et al. 2003). No enzymatic modification of any of
the pesticides was detected with ligninolytic enzymes
(ligninperoxidase, manganese peroxidase, laccase) in the ex-
tracellular fraction, while the microsomal fraction was able to
transform three pesticides. The structure of degradation prod-
ucts, supported by specific inhibition experiments and the
stringent requirement for NADPH during the in vitro assays,
suggested the involvement of a CYP450 (Jauregui et al. 2003).
Another set of in vitro experiments with P. ostreatus was
carried out to track the degradation mechanisms involved in
the degradation of the synthetic hormone 17 alpha-
ethinylestradiol (EE2) (Kesinov et al. 2012). The white rot
is able to completely remove EE2 from a liquid complex or
mineral medium within 3 or 14 days, respectively. The results
documented the involvement of various simultaneous mech-
anisms in the EE2 degradation by P. ostreatus, including both
the ligninolytic system and the eukaryotic machinery of
CYP450s. EE2 was degraded by the isolated laccase of P.
ostreatus, by the intracellular microsomal fraction, and also
by a laccase-like activity associated with fungal mycelium.
The degradation was completely suppressed in the presence of
CYP450 inhibitors, piperonylbutoxide and carbon monoxide,
indicating the role of this monooxygenase in the degradation
process.
CYP450 was also detected in the microsomal fraction of
Irpex lacteus (Cajthaml et al. 2008). Several novel intermedi-
ates of PAHs degradation, probably connected with the par-
ticipation of CYP450 in their biodegradation, were detected in
this study. Nevertheless, using PAHs as substrates, no
CYP450 activity was detected in microsomal or cytosolic
fractions regardless of the culture conditions (Cajthaml et al.
2008). Covino et al. (2010) studied in vivo and in vitro PAHs
degradation by Lentinus tigrinus CBS 577.79. The
10264 Appl Microbiol Biotechnol (2013) 97:1026310273
Table 1 Implication of microsomal enzymes in xenobiotic degradation
Organism/enzyme Pollutant Note References
P. chrysosporium/
microsomal and
cytosolic fractions
Benzo(a)pyrene CYP450 and CYP 450-mediated benzo(a)pyrene
hydroxylase were detected in microsomal fractions;
benzo(a)pyrene hydroxylation was NADPH
dependent and inhibited by CO
Masaphy et al. (1996a)
P. pulmonarius /
mycelial fractions
Atrazine Increase in CYP450 concentration during atrazine
degradation; piperonyl butoxid inhibited atrazine
transformation by fungal mycelium
Masaphy et al. (1996b)
P. chrysosporium liquid
cultures/mycelial
fractions
Lindane, atrazine Microsomal CYP450 was detected in the
mycelial fractions; 1-aminobenzotriazole
reduced pesticide metabolism
Mougin et al.
(1996,1997)
P. chrysosporium
liquid cultures
Nonylphenol 100 % degradation of 100 ppm nonylphenol
by fungal cultures; degradation inhibited by
piperonyl butoxide, a CYP450 inhibitor
Subramanian and
Yadav (2009)
P. chrysosporium /
microsomal fractions
PCP PCP oxidation by microsomal fractions of the
fungus; carbon monoxide difference spectra
indicated induction of CYP450 by PCP
Ning and
Wang (2012)
P. chrysosporium
liquid cultures
Biphenyl, dibenzofuran,
diphenyl ether
Involvement of CYP450s in degradation
hydroxylation reactions on the aromatic
ring was inhibited by piperonyl butoxide
Hiratsuka et al. (2005)
P. chrysosporium
liquid cultures
Endosulfan The fungus utilizes both oxidative and hydrolytic
pathways for metabolism of endosulfan;
piperonyl butoxide inhibited the oxidation
of endosulfane and enhanced its hydrolysis
Kullmann and
Matsamura (1996)
P. chrysosporium
liquid cultures
Nitroaromatic compounds
(4-nitrotoluene,
4-nitrobenzoic acid,
4-nitrophenol)
Fungal formation of 4-nitrobenzyl alcohol and
1,2-dimethoxy-4-nitrobenzene was inhibited
by piperonyl butoxide, a CYP450 inhibitor
Teramoto et al.
(2004a,2004b)
P.chrysosporium
CYP450s (PcCYPs)
Dioxins Screening of individual PcCYPs for transformation
of dioxins; microsomal PcCYP11a3 has the
highest activity and catalyzes hydroxylation
of 2,3-dichlorodibenzo-p-dioxin
Kasai et al. (2010)
P. chrysosporium /cytochrome
P450 monooxygenases
PAHs Identification and functional characterization
of PAH-degrading CYP450 monooxygenases;
identification of 6 PAH-responsive P450
genes (Pc-pah1-Pc-pah6)
Syed et al. (2010)
P.chrysosporium /
CYP5136A3
PAHs, endocrine-disrupting
alkylphenols
CYP5136A3, cytochrome P450 monooxygenase
showed common responsiveness and catalytic
versatility towards endocrine-disrupting
alkylphenols and PAHs
Syed et al. (2011)
C. elegans /cytosolic and
microsomal fractions
PAHs, pharmaceutical
drugs
Microsomal fractions contained cytochrome P450
monooxygenase activities for aromatic hydroxylation
and N-demethylation of cyclobenzaprine
Zhang et al. (1996)
B. adusta/microsomal fractions TNT Microsomal fraction of cell grown in the
presence of TNT was found to contain CYP450;
in cells grown without TNT, no microsomal
CYP450 could be found; piperonyl butoxide
diminished TNT mineralization; TNT metabolites
were identified as aminodinitrotoluenes
Eilers et al. (1999)
P. ostreatus/mycelial extracts Phenanthrene Cytochrome P450 monooxygenase and epoxide
hydrolase were involved in the initial oxidation
of phenanthrene to form phenanthrene
trans-9,10-dihydrodiol
Bezalel et al. (1997)
P. ostreatus/microsomal
fractions
Five pesticidestrichlorfon,
phosmet, terbufos, azinphos-
methyl, malathion
The microsomal fraction was able to transform three
pesticides (phosmet, terbufos, azinphos-methyl)
Jauregui et al. (2003)
P. ostreatus/laccase,
microsomal fractions
17 alpha-ethinylestradiol (EE2) EE2 was degraded by both isolated laccase
and microsomal fractions containing
CYP450; EE2 degradation was suppressed
by piperonyl butoxide and CO
Kesinov et at. (2012)
I. lacteus liquid cultures PAHs PAHs removal by liquid fungal cultures in
a complex medium; CYP450 detected in
microsomal fractions of the fungus
Cajthaml et al. (2008)
Appl Microbiol Biotechnol (2013) 97:1026310273 10265
identification of degradation products showed the presence of
several PAH derivatives, such as quinones, dicarboxylated,
and ring fission derivatives, presumably derived from the
action of lignin-modifying enzymes. On the other hand, the
presence of hydroxylated derivatives of anthrone and phenan-
threne 9,10- dihydrodiol suggested the possible involvement
of CYP450 epoxide hydrolase system, documenting the in-
volvement of various simultaneous degradation mechanisms
similar to P. ostreatus.
Prieto et al. (2011) studied degradation of antibiotics by the
white rot fungus Trametes versicolor. More than 90 % of
ciprofloxacin (CIPRO) and norfloxacin (NOR) were degraded
Table 1 (continued)
Organism/enzyme Pollutant Note References
L. tigrinus liquid cultures/
CYP450-epoxide
hydrolase system
PAHs PAH degradation superior in shaken cultures
(up to 97 %) compared to static cultures
(<50 %); the presence of hydroxylated
derivatives suggested the involvement
of CYP450-epoxide hydrolase system
Covino et al. (2010)
T. versicolor liquid cultures Ciprofloxacin and
norfloxacin
>90 % degradation in 7 days; degradation
was inhibited by 1-aminobenzotriazole
Prieto et al. (2011)
Fusarium moniliforme/
cell extracts
Propylbenzene Hydroxylation of propylbenzene needed
molecular oxygen and NADPH, FAD, and
FMN as coenzymes; it was inhibited by CO
Uzura et al. (2001)
T. trogii, T. hirsuta,
P. chrysosporium,
T. versicolor, T. palustris
liquid cultures
Dibenzyl sulfide 1-Aminobenzotriazole eliminated
dibenzyl sulfoxide oxidation
Van Hamme
et al. (2003)
Phlebia brevispora
liquid cultures
Dieldrin Transformation included 9-hydroxylation;
a potential involvement of microsomal
monooxygenases was suggested
Kamei et al. (2010)
P. ostreatus, I. lacteus,
B. adusta, D. squalens,
P. chrysosporium, P.
magnoliae, P. cinnabarinus,
T. versicolor
PCBs - Delor 103 Degradation by liquid fungal cultures; the
involvement of intracellular enzymes
(CYP450, aryl-alcohol dehydrogenase,
aryl-aldehyde dehydrogenase) in the
degradation was suggested
vanarov
et al. (2012)
A. terreus /cytochrome P450
monooxygenases
Alkanes, alkane derivatives,
alcohols, aromatic
compounds, organic
solvents, steroids
In vitro degradation by microsomal fractions;
inhibition by taxifolin; determination of
CYP450 substrate specificity
Vatsyayan
et al. (2008)
R. nigricans/NADPH-
cytochrome P450
reductase
Progesterone NADPH-cytochrome P450 reductase is
involved in hydroxylation of progesterone
at 11alpha position; CPR was isolated
from induced mycelia and characterized
Makovec and
Breksvar (2002)
P. chrysosporium liquid cultures/
microsomal fractions
Benzoic acid CYP450-mediate degradation of benzoic acid;
induction of CYP450 by benzoic acid
Ning et al. (2010b)
P. chrysosporium liquid
cultures/microsomal
fractions
Phenanthrene Transformation of phenanthrene to
phenanthrene trans-9,10-dihydrodiol
was inhibited by piperonyl butoxide
Ning et al. (2010a)
P.chrysospporium
CYP450s (PcCYPs)
Anthracene 12 cytochrome P450 monooxygenases involved
in anthracene metabolism were identified
by transcriptomic profiling; 14 PcCYP
species catalyze stepwise conversion
of anthracene to anthraquinon via
intermediate formation of anthrone
Chigu et al. (2010)
Trichoderma harzianum
CYP450
n-Alkanes A microsomal, n-alkane-inducible CYP450
was identified; CYP450-dependent conversion
of alkanes to fatty acids allowing their
incorporation into lipids was suggested
Del Carratore
et al. (2011)
P.chrysosporium /PcCYP1f Benzoic acid Recombinant PcCYP1f catalyzed hydroxylation
of benzoic acid to 4-hydroxybenzoic acid;
PcCYP1f was induced at a transcriptional
level by benzoic acid
Matsuzaki and
Wariishi (2005)
P.chrysosporium /CYP63A3 PAHs, alkanes, oxygenated
mono aromatics
The expression of CYP63A3 was induced
by various xenobiotics
Doddapaneni
et al. (2005)
10266 Appl Microbiol Biotechnol (2013) 97:1026310273
after 7 days. Inhibition of CIPRO and NOR degradation by
the CYP450 inhibitor 1-aminobenzotriazole suggested that
the CYP450 system also played a role in the degradation of
the two antibiotics. Moreover, transformation products of
CIPRO and NOR were monitored in this study. CYP450-
mediated reaction mechanisms were also proposed for xenobi-
otic transformation in several other fungi (Uzura et al. 2001; Van
Hamme et al. 2003; Kamei et al. 2010; vanarov et al. 2012).
Abroad substrate P450 monooxygenase activity was found
in the cells of Aspergillus terreus (Vatsyayan et al. 2008). For
the first time in this study, evidence was brought for a shift in
CYP450 activity localization during biodegradation. The
P450 monooxygenase activity was localized in the cytosol
of n-hexadecane-grown cells, while it was apparently distrib-
uted in light mitochondrial and microsomal fraction of
glucose-grown cells. The substrate specificities of CYP450
present in all the locations, however, were similar irrespective
of the substrates used for the growth. Apart from cytosolic
CYP450s, the microsomal enzymes may also cooperate with
other intracellular enzymes in xenobiotic metabolism. Epox-
ide hydrolase, glutathione S-transferase, methyl transferase,
aryl-alcohol dehydrogenase, and aryl-aldehyde dehydroge-
nase activities are discussed in this view in some works
(Bezalel et al. 1997; vanarov et al. 2012; Kesinov et al.
2012). Kulmann and Matsumura (1996) suggested that P.
chrysosporium utilizes two divergent pathways for metabo-
lism of pesticide endosulfan, one hydrolytic and the other
oxidative that is catalysed by CYP450.
Microsomal enzymes-mediated biotransformation
Apart from biodegradation, microsomal proteins are also con-
nected with other biotechnologically relevant reactions in
fungi. For example, OrdA enzyme, a microsomal enzyme of
Aspergillus parasiticus, was shown to be involved in aflatoxin
biosynthesis by this fungus (Zeng et al. 2011; Yabe et al.
2012). Another step in the aflatoxin biosynthesis has been
already previously shown to be catalysed by a P450
monooxygenase encoded by the cypA gene (Ehrlich et al.
2004). Microsomal fractions of Pleurotus sapidus were used
for the conversion of alpha-pinene to verbenols, verbenone,
and minor volatile flavors (Krings et al. 2009). A highly
st er eospeci f i c monot er penol dehydr ogenase and
dioxygenases were proposed to catalyse the bioconversion
of terpene substrates in the addition to previously assumed
CYP450 enzymes (Krings et al. 2009). The microbial bio-
transformation of readily available terpenoids, like verbenone,
into more valuable compounds has economic potential in the
perfumery, food, and pharmaceutical industries. Similarly,
two strains of Aspergillus and P. digitatum have been recently
reported to hydroxylate verbenone to 10-hydroxyverbenone
(Yildirim 2011).
Further, fungal biotransformation models are also consid-
ered to be complementary sources for the preparation of
human drug metabolites that are, in many cases, critical for
further pharmacokinetics, pharmacologic, and toxic evalua-
tion of the remedy (Yang et al. 2012; Hilario et al. 2012). An
antihistamine, cyproheptadine hydrochloride, was extensively
transformed by the zygomycete C. elegans via aromatic
hydroxylation metabolic pathways (Zhang et al. 1997).
CYP450 was detected in the microsomal fractions of the
fungus and assumed to play a role in cyproheptadine hydro-
chloride metabolism. Next to bacterial CYP450 enzymes
(Otey et al. 2006) and fungal peroxygenases (Poraj-Kobielska
et al. 2011), fungal CYP450s could represent a potential
approach for human drug metabolite preparation.
An alternative genetic approach to the production of poly-
unsaturated fatty acids (PUFA) may target another group of
microsomal enzymes, fatty acid desaturases. A gene for a
microsomal delta12-fatty acid desaturase was recently isolated
from a marine alga, Pinguiochrysis pyriformis, and expressed
in yeasts and thraustochytrids that are known to accumulate
PUFA in their lipid droplets (Matsuda et al. 2011). With the
increasing demand of obtaining PUFAs from alternative
sources, the genes and enzymes involved in the biosynthesis
of PUFAwith nutraceutical potentials have been studied also
in fungi (Zhang et al. 2013; Huang et al. 2011; Sakuradani
et al. 2008). Tan et al. (2011) analyzed delta 6 desaturase and
delta 6 elongase from Conidiobolus obscurus. A novel fatty
acid elongase with wide substrate specificity was also identi-
fied in an arachidonic acid-producing fungus Mortierella
alpina 1S-4 (Sakuradani et al. 2009). However, the molecular
mechanism for functions of these enzymes is still unclear. Old
mutant and immunochemical studies described only the in-
volvement of cytochrome-b5 in fatty acid desaturation by
yeast microsomes (Ohba et al. 1979; Tamura et al. 1976).
Enzymes in fungal microsomes
Biodegradation reactions carried out by microsomal fractions
of fungi and some of the fungal biotransformations are fre-
quently connected with fungal CYP450 systems. Fungal
CYP450s catalyze the monooxygenation of a variety of hy-
drophobic substrates and are key enzymes in fungal primary
and secondary metabolisms. By the action of CYP450s, lipo-
philic compounds are metabolized to more hydrophilic deri-
vates by introducing an oxygen atom originating from molec-
ular oxygen. Typical fungal microsomal CYP450 systems are
membr ane- bound enzymes and consi st of P450
monooxygenases that primarily obtain electrons from the
P450 reductases. Beside that, electron transfer from NADH
to P450 monooxygenase via cytochrome b5-containing redox
pathways is also known (Hannemann et al. 2007; renar and
Petri 2011; Ichinose and Wariishi 2012). Most eukaryotic
Appl Microbiol Biotechnol (2013) 97:1026310273 10267
membrane-bound CYP450s are likely to have an N-terminal
TMD sequence that acts as a membrane anchor. The TMD-
associated subcellular localization to membranes should be
important for proteinprotein interactions of P450
monooxygenases with the membrane-anchored redox partners
(Nazir et al. 2010).
In membrane systems like in microsomes, a limiting
amount of P450 reductase may be effectively limiting a
P450 reaction. As a result of that, a biphasic reduction of
CYP450 is usually observed in microsomes (Guengerich
2001). Filamentous fungi with numerous CYP450s often
possess multiple microsomal redox partners, cytchrome
P450 reductases, which may also influence the specificity of
P450 monooxygenase-mediated reactions. In the plant-
pathogenic ascomycete Cochliobolus lunatus, two P450 re-
ductase paralogues, CPR1 and CPR2, supported CYP450
activity, but with different product specificities during degra-
dation of phenolic plant compounds (Lah et al. 2011). It was
concluded that CPR1 is important in primary metabolism,
whereas CPR2 plays a role in xenobiotic detoxification.
Recently, whole genome sequence analyses have revealed
large-scale divergences in basidiomycetous CYP450s, which
implies that basidiomycetes have diversified monooxygenase
functions to acquire metabolic adaptations such as xenobiotic
degradation (reviewed in Ichinose (2013)).
A high diversity of fungal CYP450 enzymes is well
reflected in genes encoding CYP450s. Fungal Cytochrome
P450 Database archives CYP450 genes in the genomes of 70
fungal species (Park et al. 2008). In P. chrysosporium,
CYP450-encoding genes were found to be differentially ex-
pressible, reflecting their functional diversity (Doddapaneni
and Yadav 2005). Despite being members of tandem gene
clusters, the genes are independently regulated and inducible
by various xenobiotics. The genes encoding P450
monooxygenases CYP63A1, A2, A3, and PcCYP1f were
shown to be inducible at a transcriptional level by certain
aliphatic hydrocarbons, oxygenated mono aromatics and low-
er molecular weight PAHs (Doddapaneni et al. 2005;
Matsuzaki and Wariishi 2005). In the case of CYP63A1 and
PcCYP1f, up-regulation of protein production in response to
benzoic acid was observed using two-dimensional electropho-
resis (Matsuzaki et al. 2008). The regulation of expression of
the family of P450 monooxygenases, the CYP63 family, in P.
chrysosporium was also studied by Subramanian and Yadav
(2008) upon induction with 42 different xenobitics. The
CYP450 genes Pff311b and Pff4a showed high levels of
induction in P. chrysosporium cultures degrading
nonylphenol (Subramanian and Yadav 2009). More recently,
an induction of microsomal CYP450s by phenanthrene,
benzoic acid, chlorbenzoic acids, and n-hexane was indicated
by carbon monoxide difference spectra analysis during the
biodegradation studies (Ning et al. 2010a,b). Twelve P.
chrysosporium P450 monooxygenases were up-regulated at
a level of transcription in response to exogenous addition of
anthracene (Chigu et al. 2010). Syed et al. (2010) identified
six PAH-responsive genes encoding P450 monooxygenases
in P. chrysosporium that were capable of PAHs oxidation.
One of them, CYP5136A3, showed a common responsive-
ness and oxidizing capability towards PAHs and endocrine-
disrupting alkylphenols (Syed et al. 2011), demonstrating the
catalytic versatility of fungal microsomal CYP450s.
Similarly to P. chrysosporium, diverse CYP450 enzyme-
encoding genes and xenobiotic-responsive CYP450 enzymes
were observed also in other filamentous fungi, like Aspergillus
oryzae (Nazir et al. 2010), A. niger (Van den Brink et al.
1996), Rhizopus nigricans (Kunic et al. 2001), and
Trichoderma harzianum (Del Carratore et al. 2011). In the
case of R. nigricans, the first strong indication that the bio-
logical role of CYP450(11alpha) induction is in detoxification
of steroids was brought by Breskvar et al. (1995) who studied
the toxic effects of steroids on fungal growth. NADPH-
cytochrome P450 reductase from R. nigricans (Makovec
and Breskvar 1998) and progesterone-induced microsomal
fungal monoxygenase system (Makovec and Breskvar 2000)
were isolated and characterized later. Compared to that, recent
fungal genome analyses projects have enabled the annotation
of many novel CYP450s, many of which are with novel
catalytic functions.
With the advancement of molecular cloning and genome
sequencing technologies, many novel fungal front-end
desaturases for the production of PUFA with nutraceutical
potentials were also described, and the enzymes were func-
tionally characterized (Zhang et al. 2013; Meesapyodsuk and
Qiu 2012; Tan et al. 2011; Zhang et al. 2007; Hongsthong
et al. 2006). These enzymes belong to membrane-bound non-
heme iron-containing oxygenases, catalyzing the formation of
a double bond in a hydrocarbon chain. Fungal front-end
desaturases are modular proteins containing a cytochrome
b5-like domain at the N-terminus. The regions of two-
membrane-spanning helices and C-terminus are probably im-
portant for the substrate selectivity and high regioselectivity of
the enzymes (Meesapyodsuk and Qiu 2012).
Despite the cited studies, our understanding of the individ-
ual functional domains of front-end desaturases still remains
limited. Being membrane-bound, this type of desaturase is
recalcitrant to biochemical purification, and therefore there is
also no information available on the 3D structure of
desaturases so far. Correspondingly to desaturases, the mem-
brane location also makes structural modelling of microsomal
CYP450 enzymes less successful compared to cytosolic ones
when extending the known P450 structural paradigm for new
enzymes (Hasemann et al. 1995). Unlike the CYP450s,
however, very little is known about the regulation of
expression of fungal front-end desaturases. All of these
findings document lacks and difficulties in the work
with membrane enzymes.
10268 Appl Microbiol Biotechnol (2013) 97:1026310273
Proteomic studies of microsomal proteins
Several individual proteins/enzymes have been isolated so far
from microsomal fractions of filamentous fungi for their func-
tional characterization (Machida and Saito 1993; Makovec
and Breskvar 1998; Maspahy et al. 1999; Makovec and
Breskvar 2000; Yoshida et al. 2000; Faber et al. 2001) or their
microsomal localization has been proven (Husson et al. 1998).
The biochemistry of microsomal cytochromes of fungi was
studied in order to develop azole antifungal agents selective
for fungi (reviewed in Yoshida (1988)). For this project,
reconstituted enzyme systems consisting of purified yeast
CYP450 enzymes were later developed and used for kinetic
analysis of the enzymes (Aoyama et al. 1991).
With the use of advanced protein analyses, the amount of
known and characterized microsomal proteins has dramatical-
ly increased, which enabled functional studies of microsomes
at the organelle level. Microsomal membrane fractions of P.
chrysosporium were analyzed to study the nature and regula-
tion of the membrane-associated components (Shary et al.
2008). Tryptic digests of the microsomal proteins were ana-
lyzed by shotgun liquid chromatographytandem mass spec-
trometry, and the results were compared against the predicted
proteome of the fungus. The resulting data sets comprised
typically 300 to 400 proteins in this study. Catalase, involved
in H
2
O
2
metabolism, and a protein belonging to glucose
methanolcholine oxidoreductase superfamily were connect-
ed with ligninolytic conditions. Microsomal preparations also
contained six proteins that could have a transporter function
and six CYP450s out of 150 encoded in the genome (Shary
et al. 2008).
Shotgun proteomics was also applied to identify the micro-
somal components involved in protein secretion by A. niger
(DeOliveira et al. 2010). Better understanding of the protein
secretion components could help to overcome the observed
limitations in protein secretion by filamentous fungi as sum-
marized in the work of Gouka et al. (1997). Proteins from the
microsomal fungal fractions of A. niger were first separated
by SDS-PAGE and trypsin-digested. After that, proteins were
analyzed by LC-MS/MS. Out of all detected proteins, 254
were predicted to play direct roles in membrane traffic and
protein secretion (DeOliveira et al. 2010). Next, this study
clearly demonstrated that D-xylose induction led to 20S pro-
teasome recruitment to the microsomal fraction and to an
increase in specific small GTPases known to be associated
with polarized growth.
In total, 1,126 microsomal proteins were identified in A.
niger microsomal protein composition resulting from cultures
grown in the conditions of amylolytic and xylanolytic enzyme
secretion (DeOliveira et al. 2011). The proteins were grouped
in the following categories: membrane traffic and protein
secretion (23 %), mitochondrial (13 %), translation (12 %),
metabolism and defense against reactive oxygen species
(12 %), cargo proteins (8 %), lipid biosynthesis (8 %), trans-
porters (5 %), and others (14 %). Similarly to the previous
work of the group (DeOliveira et al. 2010), induction of
extracellular enzyme production resulted in specific changes
in the secretory subproteome of A. niger. An association of
20S core of the proteasome with secretory organelles was also
observed in both studies, suggesting that the recruitment of the
proteasome may be a general feature of the shift to a secretion
state of the cell. Other microsomal proteins that were highly
expressed included the ribosomal assembly protein, a small
GTPase RhoA, a plasma membrane H
+
-ATPase for cell po-
larity PmaA, and a metabolic enzyme oxaloacetate acetyl
hydrolase.
In addition to the previous works, the presence of signal
sequences was predicted in the microsomal protein dataset
(DeOliveira et al. 2011). It showed that only 25 % of A. niger
microsomal proteins contained either a signal peptide or a
signal anchor. In A. niger, approximately 10 % of the total
proteins (Braaksma et al. 2010) and 92 % of the secreted
proteins (DeOliveira et al. 2011) are predicted to contain a
signal sequence. Similarly to that, only 41 % of microsomal
proteins isolated from K562 cells were assigned as membrane
proteins based on the presence of transmembrane regions or
post-translational modifications that could account for mem-
brane association (Ghosh et al. 2008). It indicates that there
may be a significant component of non-integral proteins with-
out any signal information associated with fungal microsomes.
Likewise in other organisms (Jacobs et al. 2006; Kislinger
et al. 2006; tefani et al. 2006; Ghosh et al. 2008), the cited
studies (Shary et al. 2008; DeOliveira et al. 2010; DeOliveira
et al. 2011) demonstrated the power of shotgun proteomic
analysis in the study of specific organelle fraction composition
in fungi. Proteomic studies can extend genome and tran-
scriptome analyses of fungi and fungal processes like protein
secretion. A weakness lies in the comparison of protein rela-
tive amounts in the fungal secretomes and the microsomal
proteomes based on the calculations of normalized spectral
abundance factors of proteins (DeOliveira et al. 2010;
DeOliveira et al. 2011). Because of the factor time and due
to the experimental setup of the works, the secreted proteins
accumulated over a time period. The microsomal proteome,
on the other hand, was the result of microsomes isolated in a
defined moment in time.
In comparison to the shotgun proteomic approach, two-
dimensional gel electrophoresis (2-DE)-based analyses of
subcellular membrane organelles has a disadvantage in its
low performance in the separation and analysis of membrane
proteins. Although proteins in cytoplasmic membrane
fractions of bacteria have been successfully analysed
by 2-DE (e.g., in Zuobi-Hasona et al. 2005; Petrkov
et al. 2010), the proteomic analysis of the subcellular organ-
elles containing highly hydrophobic membrane proteins re-
mains a major challenge.
Appl Microbiol Biotechnol (2013) 97:1026310273 10269
Different methods for sample preparation were tested so far
for 2-DE analyses of proteins of microsomal fractions of
plants, rat brain microsomes, and rat hepatic microsomes,
mitochondria, and endoplasmic reticulum. In most studies,
membrane samples are lysed in the presence of 14 %
CHAPS (Thomas et al. 2013; Messina et al. 2010; Galeva
and Altermann 2002; Koen and Hanzlik 2002). A combina-
tion of 4 % CHAPS and 0.5 % Triton X-100 was successfully
applied for sample lyses by Sandoval et al. (2013). In the work
of Meisrimler and Luthje (2012), sample preparations by
TCA/acetone and methanol/chloroform precipitation, with
and without SDS pre-solubilization, were compared for mi-
crosomal fractions of plant leaves and roots, showing the
superiority of methanol/chloroform precipitation and off-gel
fractionation of proteins. To improve the subsequent protein
identification, a combination of 1-DE and 2-DE-based ap-
proaches was suggested (Galeva and Altermann 2002; Koen
et al. 2013).
With all the methodical progress, the application of 2-DE
could help in the identification of differentially regulated
proteins and thus would bring additional information to shot-
gun analyses of microsomal proteomes. To our knowledge,
however, no 2-DE analyses of fungal microsomal proteins
have been published up to now.
Conclusions
Except extracellular enzymes, the biodegradation reactions per-
formed by many filamentous fungi are also assisted by intracel-
lular enzyme machinery. Microsomal CYP450s were especially
shown to metabolize a wide range of xenobiotic chemicals and
were demonstrated to be inducible by the compounds. In addi-
tion to biodegradation, the intracellular membrane-bound en-
zymes play their roles in other biotransformations, like aflatoxin
synthesis, bioconversion of verbenols, preparation of human
drug metabolites, and production of PUFA.
However, little is known about the regulation of expression
of microsomal proteins. A differential expression of most
studied enzymes, CYP450s has been extensively studied in
the model fungus P. chrysosporium only. Regulation mecha-
nisms involved in the expression of membrane-bound fatty
acid desaturases have been only hypothesized. Therefore,
further research on the composition of microsomal compo-
nents is needed to extend our understanding of the intracellu-
lar processes during the biodegradation and biotransformation
reactions in fungi.
As summarized here, the advanced protein analyses repre-
sent a high-throughput method for the identification of large
sets of proteins and thus enable the study of specific organelle
fraction composition and of the role of organelles in fungal
processes. They can extend genome and transcriptome analy-
ses of fungi.
Acknowledgments This work was supported by the projects DAAD
A/13/07824, TE01020218 of the Czech Technology Agency and the
Institutional Research Concept RVO: 61388971.
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