Microsomal fraction of fungal cells contains enzymes that play a role in biotechnologically relevant processes. Microsomal enzymes, namely, CYP450s, were shown to metabolize a wide range of xenobiotic compounds. This work reviews proteins that were identified through a shotgun proteomic approach in fungal microsomes under various culture conditions.
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Original Title
Fungal Microsomes in a Biotransformation Perspective
Microsomal fraction of fungal cells contains enzymes that play a role in biotechnologically relevant processes. Microsomal enzymes, namely, CYP450s, were shown to metabolize a wide range of xenobiotic compounds. This work reviews proteins that were identified through a shotgun proteomic approach in fungal microsomes under various culture conditions.
Microsomal fraction of fungal cells contains enzymes that play a role in biotechnologically relevant processes. Microsomal enzymes, namely, CYP450s, were shown to metabolize a wide range of xenobiotic compounds. This work reviews proteins that were identified through a shotgun proteomic approach in fungal microsomes under various culture conditions.
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. 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