Fungal Microsomes in a Biotransformation Perspective

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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 arole in biotechnologically relevant processes. Microsomal en-zymes, namely, CYP450s, were shown to metabolize a widerange of xenobiotic compounds, including PAHs, PCBs, di-oxins, and endocrine disruptors, and take part in other fungalbiotransformation reactions. However, little is known about thenature and regulation of these membrane-associated reactions.Advanced proteomic and post-genomic techniques make itpossible to identify larger numbers of microsomal proteinsand thus add to a deeper study of fungal intracellular processes.In this work, proteins that were identified through a shotgunproteomic approach in fungal microsomes under various cul-ture conditions are reviewed. However, further research is stillneeded to fully understand the role of microsomes in fungalbiodegradation 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 degradationpathways. 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; Covinoet al. 2010; Prieto et al. 2011; vanarov et al. 2012;Kesinov et al. 2012). Filamentous fungi have been thenshown to possess a high diversity of CYP450 systems with abroad substrate activity (Vatsyayan et al. 2008; Lah et al.2008). Their relation to fungal metabolism of xenobioticchemicals has been reviewed previously (renar and Petri2011; Peng et al. 2008). It underlined the need of betterunderstanding of fungal intracellular processes and opened anew line for fungal biodegradation research.

    In this work, findings supporting degradation ability andbiotransformation potential of fungal microsomal enzymes aresummarized. Fungal microsomes are equivalent to subcellularmembrane fractions that are obtained from homogenized fun-gal mycelium by differential centrifugation as described byCinti 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 byenzymatic marker assays as described in Jauregui et al.(2003). Endoplasmic reticulum marker NADH-cytochrome creductase has been determined as a marker activity for micro-somal fractions.

    To clarify the nature of microsomal processes, two types ofstudies have been nowadays carried out in fungi: narrowlyfocused works on functions of individual enzymes andproteome-wide studies. For example, mechanisms involvedin the recognition of aromatic compounds by the fungalCYP450 were studied in the model fungus Phanerochaetechrysosporium (Syed et al. 2011). Contrary to that, a largenumber of microsomal proteins were identified in Aspergillusniger (DeOliveira et al. 2010; DeOliveira et al. 2011) using

    K. Svobodov (*) :H. MikeskovLaboratory of Environmental Biotechnology, Institute ofMicrobiology ASCR, v.v.i., Videnska 1083,14220 Prague, Czech Republice-mail: [email protected]

    H. MikeskovInstitute of Chemical Technology Prague,Faculty of Food and Biochemical Technology, Technick 5,160 28 Prague 6, Czech Republic

    D. PetrkovLaboratory of Cell Signalization, Institute of Microbiology ASCR,v.v.i., Videnska 1083, 14220 Prague, Czech Republic

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  • advanced proteomic techniques. Both approaches could helpto enhance the understanding of fungal intracellular processes.In this work, changes in membrane enzymes during biodeg-radation reactions and identification of microsomal proteinsare discussed to give an insight into the nature and regulationof microsomal biodegradation and biotransformation process-es. Advanced mapping of microsomal proteomes by high-throughput proteomics is especially highlighted as a usefultool 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. Ashort review is given in Table 1.

    Several biodegradation studies suggested the involvementof intracellular enzymes in the biodegradation reactions. Thework 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 insecticidelindane and herbicide atrazine by the liquid cultures ofP. chrysosporium has been drastically reduced by1-aminobenzotriazole (a CYP450 inactivator) (Mougin et al.1996; Mougin et al. 1997). Conversely, phenobarbital (a P450inducer) did not significantly increase lindane breakdown.Various inhibition studies also affirmed the implication ofP. chrysosporium CYP450s (PcCYPs) in the degradation ofnonylphenol (Subramanian and Yadav 2009) and pentachlo-rophenol (PCP) (Ning and Wang 2012), hydroxylation ofxenobiotics (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 microsomalPcCYP designated as PcCYP11a3 showed the highest activ-ity. It catalyses the hydroxylation of 2,3- dichlorodibenzo-p-dioxin and has the highest activity towards polychlorinateddioxins among the known CYP450s derived from microor-ganisms. Recently, a genome-wide gene induction strategyrevealed multiple PcCYPs responsive to individual classesof xenobiotics (Syed et al. 2010). CYP5136A3 then showeda common responsiveness and catalytic versatility towardsendocrine-disrupting alkylphenols and polycyclic aromatichydrocarbons (PAHs; Syed et al. 2011). Metabolic pathwaysof PAHs by fungal P450 monooxygenases were alreadyreviewed in the work of Peng et al. (2008).

    Next to P. chrysosporium , the potential of microsomalfractions to metabolize xenobiotics was studied in other fungi,too. Cytosolic and microsomal fractions of Cunninghamella

    elegans were assayed for activities of cytochrome P450monooxygenase, aryl sulfotransferase, glutathione S -transfer-ase, UDP-glucuronosyltransferase, and N -acetyltansferaseand connected with the physiological versatility of the fungusin the metabolism of xenobiotics (Zhang et al. 1996). Eilerset 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 mechanismsinvolved in the degradation of phenanthrene by Pleurotusostreatus . CYP450 activity was detected in both cytosolicand microsomal fractions of the fungus; however, it wasinhibited 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 theinitial oxidation of phenanthrene to form phenanthrenetrans-9,10-dihydrodiol.

    The extracellular and microsomal fractions of P. ostreatus7989 were tested for in vitro degradation of five pesticides(Jauregui et al. 2003). No enzymatic modification of any ofthe pesticides was detected with ligninolytic enzymes(ligninperoxidase, manganese peroxidase, laccase) in the ex-tracellular fraction, while the microsomal fraction was able totransform three pesticides. The structure of degradation prod-ucts, supported by specific inhibition experiments and thestringent 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 wascarried out to track the degradation mechanisms involved inthe degradation of the synthetic hormone 17 alpha-ethinylestradiol (EE2) (Kesinov et al. 2012). The white rotis able to completely remove EE2 from a liquid complex ormineral medium within 3 or 14 days, respectively. The resultsdocumented the involvement of various simultaneous mech-anisms in the EE2 degradation by P. ostreatus , including boththe ligninolytic system and the eukaryotic machinery ofCYP450s. EE2 was degraded by the isolated laccase of P.ostreatus , by the intracellular microsomal fraction, and alsoby a laccase-like activity associated with fungal mycelium.The degradation was completely suppressed in the presence ofCYP450 inhibitors, piperonylbutoxide and carbon monoxide,indicating the role of this monooxygenase in the degradationprocess.

    CYP450 was also detected in the microsomal fraction ofIrpex 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 inthis study. Nevertheless, using PAHs as substrates, noCYP450 activity was detected in microsomal or cytosolicfractions regardless of the culture conditions (Cajthaml et al.2008). Covino et al. (2010) studied in vivo and in vitro PAHsdegradation by Lentinus tigrinus CBS 577.79. The

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  • Table 1 Implication of microsomal enzymes in xenobiotic degradation

    Organism/enzyme Pollutant Note References

    P. chrysosporium/microsomal andcytosolic fractions

    Benzo(a)pyrene CYP450 and CYP 450-mediated benzo(a)pyrenehydroxylase were detected in microsomal fractions;benzo(a)pyrene hydroxylation was NADPHdependent and inhibited by CO

    Masaphy et al. (1996a)

    P. pulmonarius /mycelial fractions

    Atrazine Increase in CYP450 concentration during atrazinedegradation; piperonyl butoxid inhibited atrazinetransformation by fungal mycelium

    Masaphy et al. (1996b)

    P. chrysosporium liquidcultures/mycelialfractions

    Lindane, atrazine Microsomal CYP450 was detected in themycelial fractions; 1-aminobenzotriazolereduced pesticide metabolism

    Mougin et al.(1996,1997)

    P. chrysosporiumliquid cultures

    Nonylphenol 100 % degradation of 100 ppm nonylphenolby fungal cultures; degradation inhibited bypiperonyl butoxide, a CYP450 inhibitor

    Subramanian andYadav (2009)

    P. chrysosporium /microsomal fractions

    PCP PCP oxidation by microsomal fractions of thefungus; carbon monoxide difference spectraindicated induction of CYP450 by PCP

    Ning andWang (2012)

    P. chrysosporiumliquid cultures

    Biphenyl, dibenzofuran,diphenyl ether

    Involvement of CYP450s in degradationhydroxylation reactions on the aromaticring was inhibited by piperonyl butoxide

    Hiratsuka et al. (2005)

    P. chrysosporiumliquid cultures

    Endosulfan The fungus utilizes both oxidative and hydrolyticpathways for metabolism of endosulfan;piperonyl butoxide inhibited the oxidationof endosulfane and enhanced its hydrolysis

    Kullmann andMatsamura (1996)

    P. chrysosporiumliquid cultures

    Nitroaromatic compounds(4-nitrotoluene,4-nitrobenzoic acid,4-nitrophenol)

    Fungal formation of 4-nitrobenzyl alcohol and1,2-dimethoxy-4-nitrobenzene was inhibitedby piperonyl butoxide, a CYP450 inhibitor

    Teramoto et al.(2004a,2004b)

    P.chrysosporiumCYP450s (PcCYPs)

    Dioxins Screening of individual PcCYPs for transformationof dioxins; microsomal PcCYP11a3 has thehighest activity and catalyzes hydroxylationof 2,3-dichlorodibenzo-p-dioxin

    Kasai et al. (2010)

    P. chrysosporium /cytochromeP450 monooxygenases

    PAHs Identification and functional characterizationof PAH-degrading CYP450 monooxygenases;identification of 6 PAH-responsive P450genes (Pc-pah1-Pc-pah6)

    Syed et al. (2010)

    P.chrysosporium /CYP5136A3

    PAHs, endocrine-disruptingalkylphenols

    CYP5136A3, cytochrome P450 monooxygenaseshowed common responsiveness and catalyticversatility towards endocrine-disruptingalkylphenols and PAHs

    Syed et al. (2011)

    C . elegans /cytosolic andmicrosomal fractions

    PAHs, pharmaceuticaldrugs

    Microsomal fractions contained cytochrome P450monooxygenase activities for aromatic hydroxylationand N-demethylation of cyclobenzaprine

    Zhang et al. (1996)

    B . adusta /microsomal fractions TNT Microsomal fraction of cell grown in thepresence of TNTwas found to contain CYP450;in cells grown without TNT, no microsomalCYP450 could be found; piperonyl butoxidediminished TNT mineralization; TNT metaboliteswere identified as aminodinitrotoluenes

    Eilers et al. (1999)

    P. ostreatus/mycelial extracts Phenanthrene Cytochrome P450 monooxygenase and epoxidehydrolase were involved in the initial oxidationof phenanthrene to form phenanthrenetrans-9,10-dihydrodiol

    Bezalel et al. (1997)

    P. ostreatus/microsomalfractions

    Five pesticidestrichlorfon,phosmet, terbufos, azinphos-methyl, malathion

    The microsomal fraction was able to transform threepesticides (phosmet, terbufos, azinphos-methyl)

    Jauregui et al. (2003)

    P. ostreatus/laccase,microsomal fractions

    17 alpha-ethinylestradiol (EE2) EE2 was degraded by both isolated laccaseand microsomal fractions containingCYP450; EE2 degradation was suppressedby piperonyl butoxide and CO

    Kesinov et at. (2012)

    I . lacteus liquid cultures PAHs PAHs removal by liquid fungal cultures ina complex medium; CYP450 detected inmicrosomal fractions of the fungus

    Cajthaml et al. (2008)

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  • identification of degradation products showed the presence ofseveral PAH derivatives, such as quinones, dicarboxylated,and ring fission derivatives, presumably derived from theaction of lignin-modifying enzymes. On the other hand, thepresence 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 mechanismssimilar to P. ostreatus .

    Prieto et al. (2011) studied degradation of antibiotics by thewhite rot fungus Trametes versicolor. More than 90 % ofciprofloxacin (CIPRO) and norfloxacin (NOR) were degraded

    Table 1 (continued)

    Organism/enzyme Pollutant Note References

    L . tigrinus liquid cultures/CYP450-epoxidehydrolase system

    PAHs PAH degradation superior in shaken cultures(up to 97 %) compared to static cultures(90 % degradation in 7 days; degradationwas inhibited by 1-aminobenzotriazole

    Prieto et al. (2011)

    Fusarium moniliforme /cell extracts

    Propylbenzene Hydroxylation of propylbenzene neededmolecular oxygen and NADPH, FAD, andFMN as coenzymes; it was inhibited by CO

    Uzura et al. (2001)

    T. trogii , T. hirsuta ,P. chrysosporium,T. versicolor, T. palustrisliquid cultures

    Dibenzyl sulfide 1-Aminobenzotriazole eliminateddibenzyl sulfoxide oxidation

    Van Hammeet al. (2003)

    Phlebia brevisporaliquid cultures

    Dieldrin Transformation included 9-hydroxylation;a potential involvement of microsomalmonooxygenases 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; theinvolvement of intracellular enzymes(CYP450, aryl-alcohol dehydrogenase,aryl-aldehyde dehydrogenase) in thedegradation was suggested

    vanarovet al. (2012)

    A . terreus /cytochrome P450monooxygenases

    Alkanes, alkane derivatives,alcohols, aromaticcompounds, organicsolvents, steroids

    In vitro degradation by microsomal fractions;inhibition by taxifolin; determination ofCYP450 substrate specificity

    Vatsyayanet al. (2008)

    R . nigricans /NADPH-cytochrome P450reductase

    Progesterone NADPH-cytochrome P450 reductase isinvolved in hydroxylation of progesteroneat 11alpha position; CPR was isolatedfrom induced mycelia and characterized

    Makovec andBreksvar (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 liquidcultures/microsomalfractions

    Phenanthrene Transformation of phenanthrene tophenanthrene trans-9,10-dihydrodiolwas inhibited by piperonyl butoxide

    Ning et al. (2010a)

    P.chrysospporiumCYP450s (PcCYPs)

    Anthracene 12 cytochrome P450 monooxygenases involvedin anthracene metabolism were identifiedby transcriptomic profiling; 14 PcCYPspecies catalyze stepwise conversionof anthracene to anthraquinon viaintermediate formation of anthrone

    Chigu et al. (2010)

    Trichoderma harzianumCYP450

    n-Alkanes A microsomal, n-alkane-inducible CYP450was identified; CYP450-dependent conversionof alkanes to fatty acids allowing theirincorporation into lipids was suggested

    Del Carratoreet al. (2011)

    P.chrysosporium /PcCYP1f Benzoic acid Recombinant PcCYP1f catalyzed hydroxylationof benzoic acid to 4-hydroxybenzoic acid;PcCYP1f was induced at a transcriptionallevel by benzoic acid

    Matsuzaki andWariishi (2005)

    P.chrysosporium /CYP63A3 PAHs, alkanes, oxygenatedmono aromatics

    The expression of CYP63A3 was inducedby various xenobiotics

    Doddapaneniet al. (2005)

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  • after 7 days. Inhibition of CIPRO and NOR degradation bythe CYP450 inhibitor 1-aminobenzotriazole suggested thatthe CYP450 system also played a role in the degradation ofthe two antibiotics. Moreover, transformation products ofCIPRO 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; VanHamme et al. 2003; Kamei et al. 2010; vanarov et al. 2012).

    A broad substrate P450monooxygenase activity was foundin the cells of Aspergillus terreus (Vatsyayan et al. 2008). Forthe first time in this study, evidence was brought for a shift inCYP450 activity localization during biodegradation. TheP450 monooxygenase activity was localized in the cytosolof n -hexadecane-grown cells, while it was apparently distrib-uted in light mitochondrial and microsomal fraction ofglucose-grown cells. The substrate specificities of CYP450present in all the locations, however, were similar irrespectiveof the substrates used for the growth. Apart from cytosolicCYP450s, the microsomal enzymes may also cooperate withother 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 otheroxidative that is catalysed by CYP450.

    Microsomal enzymes-mediated biotransformation

    Apart from biodegradation, microsomal proteins are also con-nected with other biotechnologically relevant reactions infungi. For example, OrdA enzyme, a microsomal enzyme ofAspergillus parasiticus , was shown to be involved in aflatoxinbiosynthesis by this fungus (Zeng et al. 2011; Yabe et al.2012). Another step in the aflatoxin biosynthesis has beenalready previously shown to be catalysed by a P450monooxygenase encoded by the cypA gene (Ehrlich et al.2004). Microsomal fractions of Pleurotus sapidus were usedfor the conversion of alpha-pinene to verbenols, verbenone,and minor volatile flavors (Krings et al. 2009). A highlystereospecif ic monoterpenol dehydrogenase anddioxygenases were proposed to catalyse the bioconversionof terpene substrates in the addition to previously assumedCYP450 enzymes (Krings et al. 2009). The microbial bio-transformation of readily available terpenoids, like verbenone,into more valuable compounds has economic potential in theperfumery, food, and pharmaceutical industries. Similarly,two strains ofAspergillus and P. digitatum have been recentlyreported to hydroxylate verbenone to 10-hydroxyverbenone(Yildirim 2011).

    Further, fungal biotransformation models are also consid-ered to be complementary sources for the preparation ofhuman drug metabolites that are, in many cases, critical forfurther pharmacokinetics, pharmacologic, and toxic evalua-tion of the remedy (Yang et al. 2012; Hilario et al. 2012). Anantihistamine, cyproheptadine hydrochloride, was extensivelytransformed by the zygomycete C . elegans via aromatichydroxylation metabolic pathways (Zhang et al. 1997).CYP450 was detected in the microsomal fractions of thefungus and assumed to play a role in cyproheptadine hydro-chloride metabolism. Next to bacterial CYP450 enzymes(Otey et al. 2006) and fungal peroxygenases (Poraj-Kobielskaet al. 2011), fungal CYP450s could represent a potentialapproach for human drug metabolite preparation.

    An alternative genetic approach to the production of poly-unsaturated fatty acids (PUFA) may target another group ofmicrosomal enzymes, fatty acid desaturases. A gene for amicrosomal delta12-fatty acid desaturase was recently isolatedfrom a marine alga, Pinguiochrysis pyriformis , and expressedin yeasts and thraustochytrids that are known to accumulatePUFA in their lipid droplets (Matsuda et al. 2011). With theincreasing demand of obtaining PUFAs from alternativesources, the genes and enzymes involved in the biosynthesisof PUFAwith nutraceutical potentials have been studied alsoin fungi (Zhang et al. 2013; Huang et al. 2011; Sakuradaniet al. 2008). Tan et al. (2011) analyzed delta 6 desaturase anddelta 6 elongase from Conidiobolus obscurus . A novel fattyacid elongase with wide substrate specificity was also identi-fied in an arachidonic acid-producing fungus Mortierellaalpina 1S-4 (Sakuradani et al. 2009). However, the molecularmechanism for functions of these enzymes is still unclear. Oldmutant and immunochemical studies described only the in-volvement of cytochrome-b5 in fatty acid desaturation byyeast microsomes (Ohba et al. 1979; Tamura et al. 1976).

    Enzymes in fungal microsomes

    Biodegradation reactions carried out by microsomal fractionsof fungi and some of the fungal biotransformations are fre-quently connected with fungal CYP450 systems. FungalCYP450s catalyze the monooxygenation of a variety of hy-drophobic substrates and are key enzymes in fungal primaryand 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 aremembrane-bound enzymes and consis t of P450monooxygenases that primarily obtain electrons from theP450 reductases. Beside that, electron transfer from NADHto P450 monooxygenase via cytochrome b5-containing redoxpathways is also known (Hannemann et al. 2007; renar andPetri 2011; Ichinose and Wariishi 2012). Most eukaryotic

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  • membrane-bound CYP450s are likely to have an N-terminalTMD sequence that acts as a membrane anchor. The TMD-associated subcellular localization to membranes should beimportant for proteinprotein interactions of P450monooxygenases with themembrane-anchored redox partners(Nazir et al. 2010).

    In membrane systems like in microsomes, a limitingamount of P450 reductase may be effectively limiting aP450 reaction. As a result of that, a biphasic reduction ofCYP450 is usually observed in microsomes (Guengerich2001). Filamentous fungi with numerous CYP450s oftenpossess multiple microsomal redox partners, cytchromeP450 reductases, which may also influence the specificity ofP450 monooxygenase-mediated reactions. In the plant-pathogenic ascomycete Cochliobolus lunatus , two P450 re-ductase paralogues, CPR1 and CPR2, supported CYP450activity, but with different product specificities during degra-dation of phenolic plant compounds (Lah et al. 2011). It wasconcluded that CPR1 is important in primary metabolism,whereas CPR2 plays a role in xenobiotic detoxification.Recently, whole genome sequence analyses have revealedlarge-scale divergences in basidiomycetous CYP450s, whichimplies that basidiomycetes have diversified monooxygenasefunctions to acquire metabolic adaptations such as xenobioticdegradation (reviewed in Ichinose (2013)).

    A high diversity of fungal CYP450 enzymes is wellreflected in genes encoding CYP450s. Fungal CytochromeP450 Database archives CYP450 genes in the genomes of 70fungal species (Park et al. 2008). In P. chrysosporium ,CYP450-encoding genes were found to be differentially ex-pressible, reflecting their functional diversity (Doddapaneniand Yadav 2005). Despite being members of tandem geneclusters, the genes are independently regulated and inducibleby various xenobiotics. The genes encoding P450monooxygenases CYP63A1, A2, A3, and PcCYP1f wereshown to be inducible at a transcriptional level by certainaliphatic hydrocarbons, oxygenated mono aromatics and low-er molecular weight PAHs (Doddapaneni et al. 2005;Matsuzaki and Wariishi 2005). In the case of CYP63A1 andPcCYP1f, up-regulation of protein production in response tobenzoic acid was observed using two-dimensional electropho-resis (Matsuzaki et al. 2008). The regulation of expression ofthe family of P450 monooxygenases, the CYP63 family, in P.chrysosporium was also studied by Subramanian and Yadav(2008) upon induction with 42 different xenobitics. TheCYP450 genes Pff311b and Pff4a showed high levels ofinduction in P. chrysosporium cultures degradingnonylphenol (Subramanian and Yadav 2009). More recently,an induction of microsomal CYP450s by phenanthrene,benzoic acid, chlorbenzoic acids, and n -hexane was indicatedby carbon monoxide difference spectra analysis during thebiodegradation studies (Ning et al. 2010a,b). Twelve P.chrysosporium P450 monooxygenases were up-regulated at

    a level of transcription in response to exogenous addition ofanthracene (Chigu et al. 2010). Syed et al. (2010) identifiedsix PAH-responsive genes encoding P450 monooxygenasesin 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 thecatalytic versatility of fungal microsomal CYP450s.

    Similarly to P. chrysosporium , diverse CYP450 enzyme-encoding genes and xenobiotic-responsive CYP450 enzymeswere observed also in other filamentous fungi, likeAspergillusoryzae (Nazir et al. 2010), A . niger (Van den Brink et al.1996), Rhizopus nigricans (Kunic et al. 2001), andTrichoderma harzianum (Del Carratore et al. 2011). In thecase of R . nigricans , the first strong indication that the bio-logical role of CYP450(11alpha) induction is in detoxificationof steroids was brought by Breskvar et al. (1995) who studiedthe toxic effects of steroids on fungal growth. NADPH-cytochrome P450 reductase from R . nigricans (Makovecand Breskvar 1998) and progesterone-induced microsomalfungal monoxygenase system (Makovec and Breskvar 2000)were isolated and characterized later. Compared to that, recentfungal genome analyses projects have enabled the annotationof many novel CYP450s, many of which are with novelcatalytic functions.

    With the advancement of molecular cloning and genomesequencing technologies, many novel fungal front-enddesaturases for the production of PUFA with nutraceuticalpotentials were also described, and the enzymes were func-tionally characterized (Zhang et al. 2013; Meesapyodsuk andQiu 2012; Tan et al. 2011; Zhang et al. 2007; Hongsthonget al. 2006). These enzymes belong to membrane-bound non-heme iron-containing oxygenases, catalyzing the formation ofa double bond in a hydrocarbon chain. Fungal front-enddesaturases are modular proteins containing a cytochromeb5-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 ofthe enzymes (Meesapyodsuk and Qiu 2012).

    Despite the cited studies, our understanding of the individ-ual functional domains of front-end desaturases still remainslimited. Being membrane-bound, this type of desaturase isrecalcitrant to biochemical purification, and therefore there isalso no information available on the 3D structure ofdesaturases so far. Correspondingly to desaturases, the mem-brane location also makes structural modelling of microsomalCYP450 enzymes less successful compared to cytosolic oneswhen extending the known P450 structural paradigm for newenzymes (Hasemann et al. 1995). Unlike the CYP450s,however, very little is known about the regulation ofexpression of fungal front-end desaturases. All of thesefindings document lacks and difficulties in the workwith membrane enzymes.

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  • Proteomic studies of microsomal proteins

    Several individual proteins/enzymes have been isolated so farfrommicrosomal fractions of filamentous fungi for their func-tional characterization (Machida and Saito 1993; Makovecand Breskvar 1998; Maspahy et al. 1999; Makovec andBreskvar 2000; Yoshida et al. 2000; Faber et al. 2001) or theirmicrosomal localization has been proven (Husson et al. 1998).The biochemistry of microsomal cytochromes of fungi wasstudied in order to develop azole antifungal agents selectivefor fungi (reviewed in Yoshida (1988)). For this project,reconstituted enzyme systems consisting of purified yeastCYP450 enzymes were later developed and used for kineticanalysis of the enzymes (Aoyama et al. 1991).

    With the use of advanced protein analyses, the amount ofknown and characterized microsomal proteins has dramatical-ly increased, which enabled functional studies of microsomesat 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 predictedproteome of the fungus. The resulting data sets comprisedtypically 300 to 400 proteins in this study. Catalase, involvedin H2O2 metabolism, and a protein belonging to glucosemethanolcholine oxidoreductase superfamily were connect-ed with ligninolytic conditions. Microsomal preparations alsocontained six proteins that could have a transporter functionand six CYP450s out of 150 encoded in the genome (Sharyet 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 proteinsecretion components could help to overcome the observedlimitations in protein secretion by filamentous fungi as sum-marized in the work of Gouka et al. (1997). Proteins from themicrosomal fungal fractions of A . niger were first separatedby SDS-PAGE and trypsin-digested. After that, proteins wereanalyzed by LC-MS/MS. Out of all detected proteins, 254were predicted to play direct roles in membrane traffic andprotein secretion (DeOliveira et al. 2010). Next, this studyclearly demonstrated that D-xylose induction led to 20S pro-teasome recruitment to the microsomal fraction and to anincrease in specific small GTPases known to be associatedwith polarized growth.

    In total, 1,126 microsomal proteins were identified in A .niger microsomal protein composition resulting from culturesgrown in the conditions of amylolytic and xylanolytic enzymesecretion (DeOliveira et al. 2011). The proteins were groupedin the following categories: membrane traffic and proteinsecretion (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 previouswork of the group (DeOliveira et al. 2010), induction ofextracellular enzyme production resulted in specific changesin the secretory subproteome of A . niger. An association of20S core of the proteasome with secretory organelles was alsoobserved in both studies, suggesting that the recruitment of theproteasome may be a general feature of the shift to a secretionstate of the cell. Other microsomal proteins that were highlyexpressed included the ribosomal assembly protein, a smallGTPase RhoA, a plasma membrane H+-ATPase for cell po-larity PmaA, and a metabolic enzyme oxaloacetate acetylhydrolase.

    In addition to the previous works, the presence of signalsequences was predicted in the microsomal protein dataset(DeOliveira et al. 2011). It showed that only 25 % of A . nigermicrosomal proteins contained either a signal peptide or asignal anchor. In A . niger, approximately 10 % of the totalproteins (Braaksma et al. 2010) and 92 % of the secretedproteins (DeOliveira et al. 2011) are predicted to contain asignal sequence. Similarly to that, only 41 % of microsomalproteins isolated from K562 cells were assigned as membraneproteins based on the presence of transmembrane regions orpost-translational modifications that could account for mem-brane association (Ghosh et al. 2008). It indicates that theremay 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; Kislingeret al. 2006; tefani et al. 2006; Ghosh et al. 2008), the citedstudies (Shary et al. 2008; DeOliveira et al. 2010; DeOliveiraet al. 2011) demonstrated the power of shotgun proteomicanalysis in the study of specific organelle fraction compositionin fungi. Proteomic studies can extend genome and tran-scriptome analyses of fungi and fungal processes like proteinsecretion. A weakness lies in the comparison of protein rela-tive amounts in the fungal secretomes and the microsomalproteomes based on the calculations of normalized spectralabundance factors of proteins (DeOliveira et al. 2010;DeOliveira et al. 2011). Because of the factor time and dueto the experimental setup of the works, the secreted proteinsaccumulated over a time period. The microsomal proteome,on the other hand, was the result of microsomes isolated in adefined moment in time.

    In comparison to the shotgun proteomic approach, two-dimensional gel electrophoresis (2-DE)-based analyses ofsubcellular membrane organelles has a disadvantage in itslow performance in the separation and analysis of membraneproteins. Although proteins in cytoplasmic membranefractions of bacteria have been successfully analysedby 2-DE (e.g., in Zuobi-Hasona et al. 2005; Petrkovet 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 farfor 2-DE analyses of proteins of microsomal fractions ofplants, 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; Galevaand Altermann 2002; Koen and Hanzlik 2002). A combina-tion of 4 % CHAPS and 0.5 % Triton X-100 was successfullyapplied for sample lyses by Sandoval et al. (2013). In the workof Meisrimler and Luthje (2012), sample preparations byTCA/acetone and methanol/chloroform precipitation, withand without SDS pre-solubilization, were compared for mi-crosomal fractions of plant leaves and roots, showing thesuperiority of methanol/chloroform precipitation and off-gelfractionation of proteins. To improve the subsequent proteinidentification, a combination of 1-DE and 2-DE-based ap-proaches was suggested (Galeva and Altermann 2002; Koenet al. 2013).

    With all the methodical progress, the application of 2-DEcould help in the identification of differentially regulatedproteins and thus would bring additional information to shot-gun analyses of microsomal proteomes. To our knowledge,however, no 2-DE analyses of fungal microsomal proteinshave 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 especiallyshown to metabolize a wide range of xenobiotic chemicals andwere 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 aflatoxinsynthesis, bioconversion of verbenols, preparation of humandrug metabolites, and production of PUFA.

    However, little is known about the regulation of expressionof microsomal proteins. A differential expression of moststudied enzymes, CYP450s has been extensively studied inthe model fungus P. chrysosporium only. Regulation mecha-nisms involved in the expression of membrane-bound fattyacid 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 biotransformationreactions in fungi.

    As summarized here, the advanced protein analyses repre-sent a high-throughput method for the identification of largesets of proteins and thus enable the study of specific organellefraction composition and of the role of organelles in fungalprocesses. They can extend genome and transcriptome analy-ses of fungi.

    Acknowledgments This work was supported by the projects DAADA/13/07824, TE01020218 of the Czech Technology Agency and theInstitutional Research Concept RVO: 61388971.

    References

    Aoyama Y, Yoshida Y, Sonoda Y, Sato Y (1991) Role of the side-chain oflanosterol in substrate recognition and catalytic activity of lanosterol14-alpha-demethylase (cytochrome-P-450(14DM) of yeast.Biochim Biophys Acta 1081(3):262266

    Bezalel L, Hadar Y, Cerniglia CE (1997) Enzymatic mechanisms in-volved in phenanthrene degradation by the white fungus Pleurotusostreatus . Appl Environ Microbiol 63(7):24952501

    Braaksma M, Martens-Uzunova ES, Punt PJ, Schaap PJ (2010) Aninventory of the Aspergillus niger secretome by combining in silicopredictions with shotgun proteomics data. BMC Genomics 11:584

    Breskvar K, Ferencak Z, Hudnik-Plevnik T (1995) The role of cyto-chrome P450(11-alpha) in detoxification of steroids in the filamen-tous fungus Rhizopus nigricans . J Steroid BiochemMol Biol 52(3):271275

    Cajthaml T, Erbanov P, Kollmann A, Novotn , aek V, Mougin C(2008) Degradation of PAHs by ligninolytic enzymes of Irpexlacteus. Folia Microbiol 53(4):289294

    Chigu NL, Hirosue S, Nakamura C, Teramoto H, Ichinose H, Wariishi H(2010) Cytochrome P450 monooxygenases involved in anthracenemetabolism by the white-rot basidiomycete Phanerochaetechrysosporium . Appl Microbiol Biotechnol 87:19071916

    Cinti DL, Moldeus P, Schenkman JB (1972) Kinetic parametrs of drug-metabolizing enzymes in Ca2+-sedimented microsomes from ratliver. Biochem Pharmacol 21:32493256

    Covino S, Svobodov K, Kesinov Z, Petruccioli M, Federici F,DAnnibale A, vanarov M, Cajthaml T (2010) In vivo andin vitro polycyclic aromatic hydrocarbons degradation by Lentinus(Panus) tigrinus CBS 277.79. Bioresour Technol 101(9):30043012

    renar B, Petri (2011) Cytochrome P450 enzymes in the fungalkingdom. Biochim Biophys Acta Proteins Proteomics 1814:2935

    vanarov M, Kesinov Z, Filipov A, Covino S, Cajthaml T (2012)Biodegradation of PCBs by ligninolytic fungi and characterizationof the degradation products. Chemosphere 88(11):13171323

    Del Carratore R, Gervasi PG, Contini MP, Beffy P, Maserti BE,Giovannetti G, Brondolo A, Longo V (2011) Expression and char-acterization of two new alkane-inducible cytochrome P450s fromTrichoderma harzianum. Biotechnol Lett 33:12011206

    DeOliveira JMPF, VanPassel MWJ, Schaap PJ, DeGraaff LH (2010)Shotgun proteomics of Aspergillus niger microsomes upon D-xy-lose induction. Appl Environ Microbiol 76(13):44214429

    DeOliveira JMPF, VanPassel MWJ, Schaap PJ, DeGraaff LH (2011)Proteomic analysis of the secretory response of Aspergillus nigerto D-maltose and D-xylose. PLoS ONE 6(6):e20865

    Doddapaneni H, Yadav JS (2005) Microarray-based global differentialexpression profiling of P450 monooxygenases and regulatory pro-teins for signal transduction pathways in the white rot fungusPhanerochaete chrysosporium . Mol Gen Genomics 274:454466

    Doddapaneni H, Subramanian V, Yadav JS (2005) Physiological regula-tion, xenobiotic induction, and heterologous expression of P450monooxygenase gene pc-3 (CYP63A3), a new member of theCYP63 gene cluster in the white-rot fungus Phanerochaetechrysosporium . Curr Microbiol 50:292298

    Ehrlich KC, Chang PK, Yu J, Cotty PJ (2004) Aflatoxin biosynthesiscluster gene cypA is required for G aflatoxin formation. ApplEnviron Microbiol 70:65186524

    10270 Appl Microbiol Biotechnol (2013) 97:1026310273

  • Eilers A, Rungeling E, Stundl UM, GottschalkG (1999)Metabolism of 2,4,6-trinitrotoluene by the white-rot fungus Bjerkandera adustaDSM 3375 depends on cytochrome P-450. Appl MicrobiolBiotechnol 53(1):7580

    Faber BW, VanGorcom RFM, Duine JA (2001) Purification and charac-terization of benzoate-para-hydroxylase, a cytochrome P450(CYP53A1), from Aspergillus niger. Arch Biochem Biophys394(2):245254

    Galeva N, AltermannM (2002) Comparison of one-dimensional and two-dimensional gel electrophoresis as a separation tool for proteomicanalysis of rat liver microsomes: cytochromes P450 and other mem-brane proteins. Proteomics 2:713722

    Ghosh D, Beavis RC, Wilkins JA (2008) The identification and charac-terization of membranome components. J Proteome Res 7(4):15721583

    Gouka RJ, Punt PJ, VanDenHondel CAMJJ (1997) Efficient productionof secreted proteins by Aspergillus : progress, limitations and pros-pects. Appl Microbiol Biotechnol 47:111

    Guengerich FP (2001) Common and uncommon cytochrome P450 reac-tions to metabolism and chemical toxicity. Chem Res Toxicol 14(6):611650

    Hannemann F, Bichet A, Ewen KM, Bernhardt R (2007) CytochromeP450 systemsbiological variations of electron transport chains.Biochim Biophys Acta Gen Subj 1770(3):330344

    Hasemann CA, Kurumbail RG, Boddupalli SS, Peterson JA, DeisenhoferJ (1995) Structure and function of cytochromes P450: a comparativeanalysis of three crystal structures. Structure 2:4162

    Hilario VC, Carrao DB, Barth T, Borges KB, Furtado NAJC, Pupo MT,de Oliveira ARM (2012) Assessment of the stereoselective fungalbiotransformation of albendazole and its analysis by HPLC in polarorganic mode. J Pharm Biomed Anal 61:100107

    Hiratsuka N, Oyadomari M, Shinohara H, Tanaka H, Wariishi H (2005)Metabolic mechanisms involved in hydroxylation reactions ofdiphenyl compounds by the lignin-degrading basidiomycetePhanerochaete chrysosporium . Bichem Eng J 23:241246

    Hongsthong A, Subudhi S, Sirijuntarut M, Kurdrid P, Cheevadhanarak S,TanticharoenM (2006) Revealing the complementation of ferredox-in by cytochrome b(5) in the Spirulina-delta(6)-desaturation reactionby N-terminal fusion and co-expression of the fungal-cytochromeb(5) domain and Spirulina-delta(6)-acyl-lipid desaturase. ApplMicrobiol Biotechnol 72(6):11921201

    Huang JZ, Jiang XZ, Xia XF, Yu AQ,Mao RY, ChenXF, Tian BY (2011)Cloning and functional identification of delta 5 fatty acid desaturasegene and its 5 -upstream region from marine fungusThraustochytrium sp. FJN-10. Mar Biotechnol 13(1):1221

    Husson F, Pagot Y, Kermasha S, Belin JM (1998) Fusariumproliferatum: induction and intracellular location of a lipoxygenase.Enzym Microb Technol 23(12):4248

    Ichinose H (2013) Cytochrome P450 of wood-rotting basidiomycetes andbiotechnological applications. Biotechnol Appl Biochem 60(1SI):7181

    Ichinose H, Wariishi H (2012) Heterologous expression and mechanisticinvestigation of a fungal cytochrome P450 (CYP5150A2): involve-ment of alternative redox partners. Arch Biochem Biophys 518:815

    Jacobs ME, DeSouza LV, Samaranayake H, Pearlman RE, Siu KW,Klobutcher LA (2006) The Tetrahymena thermophila phagosomeproteome. Eukaryot Cell 5:19902000

    Jauregui J, Valderrama B, Albores A, Vazquez-Duhalt R (2003)Microsomal transformation of organophosphorus pesticides bywhite rot fungi. Biodegradation 14:397406

    Kamei I, Takagi K, Kondo R (2010) Bioconversion of dieldrin by wood-rotting fungi and metabolite detection. Pest Manag Sci 66:888891

    Kasai N, Ikushiro S, Shinkyo R, Yasuda K, Hirosue S, Arisawa A,Ichinose H, Wariishi H, Sakaki T (2010) Metabolism of mono-and dichloro-dibenzo-p-dioxins by Phanerochaete chrysosporiumcytochromes P450. Appl Microbiol Biotechnol 86:773780

    Kislinger T, Cox B, Kannan A, Chung C, Hu P, Ignatchenko A, Scott MS,Gramolini AO, Morris Q, Hallett MT, Rossant J, Hughes TR, FreyB, Emili A (2006) Global survey of organ and organelle proteinexpression in mouse: combined proteomic and transcriptomic pro-filing. Cell 125:173186

    Koen YM, Hanzlik RP (2002) Identification of seven proteins in theendoplasmic reticulum as targets for reactive metabolites ofbromobenzene. Chem Res Toxicol 15:699706

    Koen YM, Sarma D, Hajovsky H, Galeva NA, Williams TD, StaudingerJL, Hanzlik RP (2013) Protein targets of thioacetamide metabolitesin rat hepatocytes. Chem Res Toxicol 26:564574

    Kesinov Z, Moeder M, Ezechi M, Svobodov K, Cajthaml T (2012)Mechanistic study of 17-ethinylestradiol biodegradation byPleurotus ostreatus: tracking of extracellular and intracellular deg-radation mechanisms. Environ Sci Technol 46:1337713385

    Krings U, Lehnert N, Fraatz MA, Hardebusch B, Zorn H, Berger RG(2009) Autooxidation versus biotransformation of alpha-pinene toflavors with Pleurotus sapidus : regioselective hydroperoxidation ofalpha-pinene and stereoselective dehydrogenation of verbenol. JAgric Food Chem 57(21):99449950

    Kullman SW, Matsumura F (1996) Metabolic pathways utilized byPhanerochaete chrysosporium for degradation of the cyclodienepesticide endosulfan. Appl Environ Microbiol 62(2):593600

    Kunic B, Truan G, Breskvar K, Pompon D (2001) Functional cloning,based on azole resistance in Saccharomyces cerevisiae , and charac-terization of Rhizopus nigricans redox carriers that are differentiallyinvolved in the P450-dependent response to progesterone stress.Mol Gen Genomics 265(5):930940

    Lah L, Kraevec N, Trontelj P, Komel R (2008) High diversity andcomplex evolution of fungal cytochrome P450 reductase: cyto-chrome P450 systems. Fung Genet Biol 45:446458

    Lah L, Podobnik B, NovakM, Koroec B, Berne S, VogelsangM, KraevecN, Zupanec N, Stojan J, Bohlmann J, Komel R (2011) The versatilityof the fungal cytochrome P450monooxygenase systm is instrumentalin xenobiotic detoxification. Mol Microbiol 81(5):13741389

    Machida S, Saito M (1993) Purification and characterisation ofmembrane-bound chitin synthase. J Biol Chem 268(3):17021707

    Makovec T, Breskvar K (1998) Purification and characterization ofNADPH-cytochrome P450 reductase from filamentous fungusRhizopus nigricans . Arch Biochem Biophys 357(2):310316

    Makovec T, Breskvar K (2000) Purification of cytochrome P450 fromfilamentous fungus Rhizopus nigricans . Pflugers Arch-Eur JPhysiol 439(3):R111R112

    Makovec T, Breskvar K (2002) Catalytic and immunochemical propertiesof NADPH-cytochrome P450 reductase from fungus Rhizopusnigricans . J Steroid Biochem Mol Biol 82(1):8996

    Masaphy S, Levanon D, Henis Y, Venkateswarlu K, Kelly SL (1996a)Evidence for cytochrome P450 and P450-mediated benzo(a)pyrenehydroxylation in the white rot fungus Phanerochaetechrysosporium . FEMS Microbiol Lett 135(1):5155

    Masaphy S, Henis Y, Levanon D (1996b) Manganese-enhanced biotrans-formation of atrazine by the white rot fungus Pleurotus pulmonariusand its correlation with oxidation activity. Appl Environ Microbiol62(10):35873593

    Maspahy S, Lamb DC, Kelly SL (1999) Purification and characterizationof a benzo(a)pyrene hydroxylase from Pleurotus pulmonarius .Biochem Biophys Res Commun 266(2):326329

    Matsuda T, Sakaguchi K, Kobayashi T, Abe E, Kurano N, Sato A, OkitaY, Sugimoto S, Hama Y, Hayashi M, Okino N, Ito M (2011)Molecular cloning of a Pinguiochrysis pyriformis oleate-specificmicrosomal delta12-fatty acid desaturase and functional analysis inyeasts and thraustochytrids. J Biochem 150(4):375383

    Matsuzaki F, Wariishi H (2005) Molecular characterization of cyto-chrome P450 catalyzing hydroxylation of benzoates from thewhite-rot fungus Phanerochaete chrysosporium . BiochemBiophys Res Commun 334:11841190

    Appl Microbiol Biotechnol (2013) 97:1026310273 10271

  • Matsuzaki F, Shimizu M, Wariishi H (2008) Proteomic and metabolomicanalyses of the white-rot fungus Phanerochaete chrysosporiumexposed to exogenous benzoic acid. J Prot Res 7(6):23422350

    Meesapyodsuk D, Qiu X (2012) The front-end desaturase: structure,function, evolution and biotechnological use. Lipids 47:227237

    Meisrimler CN, Luthje S (2012) IPG-strips versus off-gel fractionation:advantages and limits of two-dimensional PAGE in separation ofmicrosomal fractions of frequently used plant species and tissues. JProteom 75(9):25502562

    Messina A, Nencioni S, Gervasi PG, Gotlinger KH, Schwartzman ML,Longo V (2010) Molecular cloning and enzymatic characterizationof sheep CYP2J. Xenobiotica 40(2):109118

    Mougin C, Pericaud C, Malosse C, Laugero C, Asther M (1996)Biotransformation of the insecticide lindane by the white rot basid-iomycete Phanerochaete chrysosporium. Pestic Sci 47(1):5159

    Mougin C, Laugero C, Asther M, Chaplain V (1997) Biotransformationof s-triazine herbicides and related degradation products in liquidcultures by the white rot fungus Phanerochaete chrysosporium .Pestic Sci 49:169177

    Nazir KHMNH, Ichinose H, Wariishi H (2010) Molecular characteriza-tion and isolation of cytochrome P450 genes from the filamentousfungus Aspergillus oryzae . Arch Microbiol 192:395408

    Ning D, Wang H (2012) Involvement of cytochrome P450 in pentachlo-rophenol transformation in a white rot fungus Phanerochaetechrysosporium . PLoS ONE 7(9):e45887

    Ning D, Wang H, Ding Ch LH (2010a) Novel evidence of cytochromeP450-catalyzed oxidation of phenanthrene in Phanerochaetechrysosporium under ligninolytic conditions. Biodegradation 21:889901

    Ning D, Wang H, Zhuang Y (2010b) Induction of functional cytochromeP450 and its involvement in degradation of benzoic acid byPhanerochaete chrysosporium . Biodegradation 21:297308

    Ohba M, Sato R, Yoshida Y, Bieglmayer C, Ruis H (1979) Mutant andimmunochemical studies on the involvement of cytochrome-b5 infatty-acid desaturation by yeast microsomes. Biochim Biophys Acta572(2):352362

    Otey CR, Bandara G, Lalonde J, Takahashi K, Arnold FH (2006)Preparation of human metabolites of propranolol using laboratory-evolved bacterial cytochromes P450. Biotechnol Bioeng 93:494499

    Park J, Lee S, Choi J, Ahn K, Park B, Park J, Kang S, Lee YH (2008)Fungal cytochrome P450 database. BMC Genomics 9:402

    Peng RH, Xiong AS, Xue Y, Fu XY, Gao F, Zhao W, Tian YS, Yao QH(2008) Microbial biodegradation of polyaromatic hydrocarbons.FEMS Microbiol Rev 32:927955

    Petrkov D, emberov L, Halada P, Svoboda P, Svobodov J (2010)Stress proteins in the cytoplasmic membrane fraction of Bacillussubtilis . Folia Microbiol 55(5):427434

    Poraj-Kobielska M, Kinne M, Ullrich R, Scheibner K, Kayser G,Hammel KE, Hofrichter M (2011) Preparation of human drugmetabolites using fungal peroxygenases. Biochem Pharmacol 82:789796

    Prieto A, Moeder M, Rodil R, Adrian L, Marco-Urrea E (2011)Degradation of the antibiotics norfloxacin and ciprofloxacin by awhite-rot fungus and identification of degradation products.Bioresour Technol 102:1098710995

    Sakuradani E, Murata S, Kanamaru H, Shimizu S (2008) Functionalanalysis of a fatty acid elongase from arachidonic-producingMortierella alpina 1S-4. Appl Microbiol Biotechnol 81(3):497503

    Sakuradani E, Nojiri M, Suzuki H, Shimizu S (2009) Identification of anovel fatty acid elongase with a wide substrate specificity fromarachidonic acid-producing fungus Mortierella alpina 1S-4. ApplMicrobiol Biotechnol 84(4):709716

    Sandoval M, Luarte A, Herrera-Molina R, Varas-Godoy M, SantibanezM, Rubio FJ, Smit AB, Gundelfinger ED, Li KW, Smalla KH,Wyneken U (2013) The glycolytic enzyme aldolase C is up-

    regulated in rat forebrain microsomes and in the cerebrospinal fluidafter repetitive fluoxetine treatment. Brain Res 1520:114

    Shary S, Kapich AN, Panisko EA, Magnuson JK, Cullen D, Hammel KE(2008) Differential expression in Phanerochaete chrysosporium ofmembrane-associated proteins relevant to lignin degradation. ApplEnviron Microbiol 74(23):72527257

    tefani S, Palm D, Svard SG, Hehl AB (2006) Organelle proteomicsreveals cargo maturation mechanisms associated with Golgi-likeencystation vesicles in the early-diverged protozoan Giardialamblia . J Biol Chem 281:75957604

    Subramanian V, Yadav JS (2008) Regulation and heterologous expres-sion of P450 enzyme system components of the white rot fungusPhanerochaete chrysosporium . Enzym Microb Technol 43:205213

    Subramanian V, Yadav JS (2009) Role of P450 monooxygenases in thedegradation of the endocrine-disrupting chemical nonylphenol bythe white rot fungus Phanerochaete chrysosporium . Appl EnvironMicrobiol 75(17):55705580

    Syed K, Doddapaneni H, Subramanian V, Lam YW, Yadav JS (2010)Genome-to-function characterization of novel fungal P450monooxygenases oxidizing polycyclic aromatic hydrocarbons(PAHs). Biochem Biophys Res Commun 399:492497

    Syed K, Porollo A, Lam YW, Yadav JS (2011) A fungal P450(CYP5136A3) capable of oxidizing polycyclic aromatic hydrocar-bons and endocrine disrupting alkylphenols: role of Trp129 andLeu324. PLoS ONE 6(12):e28286

    Tamura Y, Yoshida Y, Sato R, Kumaoka H (1976) Fatty-acid desaturasesystem of yeast microsomesinvolvement of cytochrome b5-containing electron-transport chain. Arch Biochem Biophys175(1):284294

    Tan L, Meesapyodsuk D, Qiu X (2011) Molecular analysis of Delta 6desaturase and Delta 6 elongase from Conidiobolus obscurus in thebiosynthesis of eicosatetraenoic acid, an omega 3 fatty acid withnutraceutical potentials. Appl Microbiol Biotechnol 90(2):591601

    Teramoto H, Tanaka H, Wariishi H (2004a) Fungal cytochrome P450scatalyzing hydroxylation of substituted toluenes to form their hy-droxymethyl derivatives. FEMS Microbiol Lett 234:255260

    Teramoto H, Tanaka H,Wariishi H (2004b) Degradation of 4-nitrophenolby the lignin-degrading basidiomycete Phanerochaetechrysosporium . Appl Microbiol Biotechnol 66:312317

    Thomas A, Klein MS, Stevens AP, Reinders Y, Hellerbrand C, DettmerK, Gronwald W, Oefner PJ, Reinders J (2013) Changes in thehepatic mitochondrial and membrane proteome in mice fed a non-alcoholic steatohepatitis inducing diet. J Proteome 80:107122

    Uzura A, Suzuki T, Katsuragi T, Tani Y (2001) Involvement of cyto-chrome P450 in hydroxylation of propylbenzene by Fusariummoniliforme strain MS31. J Biosci Bioeng 92(6):580584

    Van den Brink JM, van den Hondel CAMJJ, van Gorcom RFM (1996)Optimization of the benzoate inducible benzoate p-hydroxylasecytochrome P450 enzyme system in Aspergillus niger. ApplMicrobiol Biotechnol 46(4):360364

    Van Hamme JD, Wong ET, Dettman H, Gray MR, Pickard MA (2003)Dibenzyl sulfide metabolism by white rot fungi. Appl EnvironMicrobiol 69(2):13201324

    Vatsyayan P, Kumar AK, Goswami P, Goswami P (2008) Broad substratecytochrome P450monooxygenase activity in the cells ofAspergillusterreus MTCC 6324. Bioresour Technol 99:6875

    Yabe K, Chihaya N, Hatabayashi H, Kito M, Hoshino S, Zeng H, Cai JJ,Nakajima H (2012) Production of M-/MG-group aflatoxins cata-lyzed by the OrdA enzyme in aflatoxin biosynthesis. Fungal GenetBiol 49:744754

    Yang M, Cheng CR, Yang JL, Guo DA (2012) Metabolite profiling andcharacterization for medicinal herbal remedies. Curr Drug Metab13(5):535557

    Yildirim K (2011) Biotransformation of ()-verbenone by some fungi. JChem Res 3:133134

    10272 Appl Microbiol Biotechnol (2013) 97:1026310273

  • Yoshida Y (1988) Cytochrome P450 of fungi: primary target for azoleantifungal agents. Curr Top Med Mycolog 2:388418

    Yoshida T, Kato Y, Asada Y, Nakajima T (2000) Filamentous fungusAspergillus oryzae has two types of alpha-1,2-mannosidases,one of which is a microsomal enzyme that removes a singlemannose residue from Man(9)GlcNAc(2). Glycoconj J17(11):745748

    Zeng HM, Hatabayashi H, Nakagawa H, Cai JJ, Suzuki R, Sakuno E,Tanaka T, Ito Y, Ehrlich KC, Nakajima H, Yabe K (2011)Conversion of 11-hydroxy-O-methylsterigmatocystin to aflatoxinG(1) in Aspergillus parasiticus. Appl Microbiol Biotechnol 90(2):635650

    Zhang DL, Yang YF, Leakey JEA, Cerniglia CE (1996) Phase I and phaseII enzymes produces by Cunninghamella elegans for the metabo-lism of xenobiotics. FEMS Microbiol Lett 138(23):221226

    Zhang DL, Hansen EB, Deck J, Heinze TM, Henderson A, KorfmacherWA, Cerniglia CE (1997) Fungal transformations of antihistamines:metabolism of cyproheptadine hydrochloride by Cunninghamellaelegans . Xenobiotica 27(3):301315

    Zhang S, Sakuradani E, Ito K, Shimizu S (2007) Identification of a novelbifunctional delta 12/delta 15 fatty acid desaturase from a basidio-mycete, Coprinus cinereus TD#822-2. FEBS Lett 581:315319

    Zhang R, Zhu Y, Ren L, Zhou P, Hu J, Yu L (2013) Identification of afatty acid a delta(6)-desaturase gene from the eicosapentaenoic acid-producing fungus Pythium splendens RBB-5. Biotechnol Lett35(3):431438

    Zuobi-Hasona K, Crowley PJ, Hasona A, Bleiweis AS, Brady LJ (2005)Solubilization of cellular membrane proteins from Streptococcusmutans for two-dimensional gel electrophoresis. Electrophoresis26:12001205

    Appl Microbiol Biotechnol (2013) 97:1026310273 10273

    Fungal microsomes in a biotransformation perspective: protein nature of membrane-associated reactionsAbstractIntroductionBiodegradation potential of fungal microsomesMicrosomal enzymes-mediated biotransformationEnzymes in fungal microsomesProteomic studies of microsomal proteinsConclusionsReferences