Conversion and synthesis of chemicals catalyzed by fungal cytochrome P450 monooxygenases: A review.

Cytochrome P450s (also called CYPs or P450s) are a superfamily of heme-containing monooxygenases. They are distributed in all biological kingdoms. Most fungi have at least two P450-encoding genes, CYP51 and CYP61, which are housekeeping genes that play important roles in the synthesis of sterols. However, the kingdom fungi is an interesting source of numerous P450s. Here, we review reports on fungal P450s and their applications in the bioconversion and biosynthesis of chemicals. We highlight their history, availability, and versatility. We describe their involvement in hydroxylation, dealkylation, oxygenation, C═C epoxidation, C-C cleavage, C-C ring formation and expansion, C-C ring contraction, and uncommon reactions in bioconversion and/or biosynthesis pathways. The ability of P450s to catalyze these reactions makes them promising enzymes for many applications. Thus, we also discuss future prospects in this field. We hope that this review will stimulate further study and exploitation of fungal P450s for specific reactions and applications.

Biochemical Characterization of CYP505D6, a Self-Sufficient Cytochrome P450 from the White-Rot Fungus Phanerochaete chrysosporium.

The activity of a self-sufficient cytochrome P450 enzyme, CYP505D6, from the lignin-degrading basidiomycete Phanerochaete chrysosporium was characterized. Recombinant CYP505D6 was produced in Escherichia coli and purified. In the presence of NADPH, CYP505D6 used a series of saturated fatty alcohols with C9-18 carbon chain lengths as the substrates. Hydroxylation occurred at the ω-1 to ω-6 positions of such substrates with C9-15 carbon chain lengths, except for 1-dodecanol, which was hydroxylated at the ω-1 to ω-7 positions. Fatty acids were also substrates of CYP505D6. Based on the sequence alignment, the corresponding amino acid of Tyr51, which is located at the entrance to the active-site pocket in CYP102A1, was Val51 in CYP505D6. To understand the diverse hydroxylation mechanism, wild-type CYP505D6 and its V51Y variant and wild-type CYP102A1 and its Y51V variant were generated, and the products of their reaction with dodecanoic acid were analyzed. Compared with wild-type CYP505D6, its V51Y variant generated few products hydroxylated at the ω-4 to ω-6 positions. The products generated by wild-type CYP102A1 were hydroxylated at the ω-1 to ω-4 positions, whereas its Y51V variant generated ω-1 to ω-7 hydroxydodecanoic acids. These observations indicated that Val51 plays an important role in determining the regiospecificity of fatty acid hydroxylation, at least that at the ω-4 to ω-6 positions. Aromatic compounds, such as naphthalene and 1-naphthol, were also hydroxylated by CYP505D6. These findings highlight a unique broad substrate spectrum of CYP505D6, rendering it an attractive candidate enzyme for the biotechnological industry.IMPORTANCEPhanerochaete chrysosporium is a white-rot fungus whose metabolism of lignin, aromatic pollutants, and lipids has been most extensively studied. This fungus harbors 154 cytochrome P450-encoding genes in the genome. As evidenced in this study, P. chrysosporium CYP505D6, a fused protein of P450 and its reductase, hydroxylates fatty alcohols (C9-15) and fatty acids (C9-15) at the ω-1 to ω-7 or ω-1 to ω-6 positions, respectively. Naphthalene and 1-naphthol were also hydroxylated, indicating that the substrate specificity of CYP505D6 is broader than those of the known fused proteins CYP102A1 and CYP505A1. The substrate versatility of CYP505D6 makes this enzyme an attractive candidate for biotechnological applications.

Reconstitution of biosynthetic machinery of fungal polyketides: unexpected oxidations of biosynthetic intermediates by expression host.

Reconstitution of whole biosynthetic genes in Aspergillus oryzae has successfully applied for total biosynthesis of various fungal natural products. Heterologous production of fungal metabolites sometimes suffers unexpected side reactions by host enzymes. In the studies on fungal polyketides solanapyrone and cytochalasin, unexpected oxidations of terminal olefin of biosynthetic intermediates were found to give one and four by-products by host enzymes of the transformants harboring biosynthetic genes. In this paper, we reported structure determination of by-products and described a simple solution to avoid the undesired reaction by introducing the downstream gene in the heterologous production of solanapyrone C.

Spatial Geometries of Self-Assembled Chitohexaose Monolayers Regulate Myoblast Fusion.

Myoblast fusion into functionally-distinct myotubes to form in vitro skeletal muscle constructs under differentiation serum-free conditions still remains a challenge. Herein, we report that our microtopographical carbohydrate substrates composed of bioactive hexa-N-acetyl-d-glucosamine (GlcNAc6) modulated the efficiency of myoblast fusion without requiring horse serum or any differentiation medium during cell culture. Promotion of the differentiation of dissociated mononucleated skeletal myoblasts (C2C12; a mouse myoblast cell line) into robust myotubes was found only on GlcNAc6 micropatterns, whereas the myoblasts on control, non-patterned GlcNAc6 substrates or GlcNAc6-free patterns exhibited an undifferentiated form. We also examined the possible role of GlcNAc6 micropatterns with various widths in the behavior of C2C12 cells in early and late stages of myogenesis through mRNA expression of myosin heavy chain (MyHC) isoforms. The spontaneous contraction of myotubes was investigated via the regulation of glucose transporter type 4 (GLUT4), which is involved in stimulating glucose uptake during cellular contraction. Narrow patterns demonstrated enhanced glucose uptake rate and generated a fast-twitch muscle fiber type, whereas the slow-twitch muscle fiber type was dominant on wider patterns. Our findings indicated that GlcNAc6-mediated integrin interactions are responsible for guiding myoblast fusion forward along with myotube formation.

High-level heterologous expression of fungal cytochrome P450s in Escherichia coli.

A thorough understanding of the sequence-structure-function relationships of cytochrome P450 (P450) is necessary to better understand the metabolic diversity of living organisms. Significant amounts of pure enzymes are sometimes required for biochemical studies, and their acquisition often relies on the possibility of their heterologous expression. In this study, we performed extensive heterologous expression of fungal P450s in Escherichia coli using 304 P450 isoforms. Using large-scale screening, we confirmed that at least 27 P450s could be expressed with/without simple sequence deletion at the 5' end of cDNAs, which encode the N-terminal hydrophobic domain of the enzyme. Moreover, we identified N-terminal amino acid sequences that can potentially be used to construct chimeric P450s, which could dramatically improve their expression levels even when the expression of the wild-type sequence was unpromising. These findings will help increase the chance of heterologous expression of a variety of fungal and other eukaryotic membrane-bound P450s in E. coli.

Thiobencarb herbicide reduces growth, photosynthetic activity, and amount of Rieske iron-sulfur protein in the diatom Thalassiosira pseudonana.

We investigated the effects of the herbicide thiobencarb on the growth, photosynthetic activity, and expression profile of photosynthesis-related proteins in the marine diatom Thalassiosira pseudonana. Growth rate was suppressed by 50% at a thiobencarb concentration of 1.26 mg/L. Growth and photosystem II activity (Fv /Fm ratio) were drastically decreased at 5 mg/L, at which the expression levels of 13 proteins increased significantly and those of 11 proteins decreased significantly. Among these proteins, the level of the Rieske iron-sulfur protein was decreased to less than half of the control level. This protein is an essential component of the cytochrome b6 f complex in the photosynthetic electron transport chain. Although the mechanism by which thiobencarb decreased the Rieske iron-sulfur protein level is not clear, these results suggest that growth was inhibited by interruption of the photosynthetic electron transport chain by thiobencarb.

Cytochrome P450 of wood-rotting basidiomycetes and biotechnological applications.

Wood-rotting basidiomycetes possess superior metabolic functions to degrade woody biomass, and these activities are indispensable for the carbon cycle of the biosphere. As well as basic studies of the biochemistry of basidiomycetes, many researchers have been focusing on utilizing basidiomycetes and/or their enzymes in the biotechnology sector; therefore, the unique activities of their extracellular and intracellular enzymes have been widely demonstrated. A rich history of applied study has established that basidiomycetes are capable of metabolizing a series of endogeneous and exogeneous compounds using cytochrome P450s (P450s). Recently, whole genome sequence analyses have revealed large-scale divergences in basidiomycetous P450s. The tremendous variation in P450s implies that basidiomycetes have vigorously diversified monooxygenase functions to acquire metabolic adaptations such as lignin degradation, secondary metabolite production, and xenobiotics detoxification. However, fungal P450s discovered from genome projects are often categorized into novel families and subfamilies, making it difficult to predict catalytic functions by sequence comparison. Experimental screening therefore remains essential to elucidate the catalytic potential of individual P450s, even in this postgenomic era. This paper archives the known metabolic capabilities of basidiomycetes, focusing on their P450s, outlines the molecular diversity of basidiomycetous P450s, and introduces new functions revealed by functionomic studies using a recently developed, rapid, functional screening system.

Growth-phase dependent variation in photosynthetic activity and cellular protein expression profile in the harmful raphidophyte Chattonella antiqua.

This study investigated temporal variations in the potential maximum quantum yield of photosystem II (F(v)/F(m) ratio) and growth-phase dependent cellular protein expressions of Chattonella antiqua under laboratory conditions. Despite the culture conditions, significant positive correlations between the F(v)/F(m) ratio and daily growth rate were observed. Threshold F(v)/F(m) ratios associated with positive cell growth were calculated to be >0.44, >0.44, and >0.37, and those associated with active cell growth (growth rate >0.5 div. d(-1)) were >0.58, >0.60, and >0.49 under control culture, low nutrient and intense light conditions, respectively. Proteome profiles obtained by two-dimensional gel electrophoresis (2-DE) indicated that 42 protein spots were differentially expressed at various growth phases of C. antiqua, which indicates changes in cellular physiological status throughout the growth cycle, and suggests that oxygen evolving enhancer 1 and 2-cysteine peroxiredoxin play roles in maintaining the positive growth of C. antiqua.

Biocatalyst collection and heterologous expression of sesquiterpene synthases from basidiomycetous fungi: Discovery of a novel sesquiterpene hydrocarbon.

Basidiomycetes produce a wide variety of sesquiterpenoids, which attract significant interest in pharmaceutical and industrial applications. Structural diversification of sesquiterpenoids is performed by sesquiterpene synthases (STSs), which produce a wide array of backbone structures; therefore, functional characterization and increased biocatalyst collection of STSs are important for expanding scientific knowledge and meeting the needs of advanced biotechnology. Gene identification and functional annotation of STSs from the basidiomycetous fungi Agaricus bisporus, Auriscalpium vulgare, Lepista nuda, Pleurotus ostreatus and Trametes versicolor were conducted. Through these investigations, the catalytic functions of 30 STSs were revealed using recombinant enzymes heterologously expressed in Saccharomyces cerevisiae. Furthermore, the unique function of an STS from P. ostreatus, PoSTS-06, was revealed to be the production of a novel sesquiterpene hydrocarbon that we named pleostene. The absolute structure of pleostene was determined by NMR spectroscopy and X-ray crystallography using the crystalline sponge method.

Molecular characterization of an insecticide-induced novel glutathione transferase in silkworm.

BACKGROUND: The glutathione transferase (GST) superfamily is involved in the detoxification of various xenobiotics. We have identified a GST mRNA that was induced in the fat bodies of a silkworm strain exhibiting diazinon resistance and have investigated the enzyme properties of this GST. METHODS: A soluble recombinant protein was overexpressed in Escherichia coli. Amino acid residues of interest were changed to alanine by site-directed mutagenesis. RESULTS AND CONCLUSIONS: Phylogenetic analysis of the deduced amino acid sequence indicates that this GST belongs to an unclassified group previously reported in mosquitoes. This enzyme, named bmGSTu, has highly conserved amino acid residues, including Tyr7, Ser12 and Asn50. A recombinant bmGSTu was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a synthetic substrate of GST. Kinetic analysis of bmGSTu mutants indicated that Tyr7, Ser12 and Asn50 are involved in enzyme function. GENERAL SIGNIFICANCE: These results support the hypothesis that bmGSTu may play a role in insecticide resistance in Bombyx mori.

Heterologous expression and mechanistic investigation of a fungal cytochrome P450 (CYP5150A2): involvement of alternative redox partners.

A fungal cytochrome P450 monooxygenase (CYP5150A2) from the white-rot basidiomycete Phanerochaete chrysosporium was heterologously expressed in Escherichia coli and purified as an active form. The purified CYP5150A2 was capable of hydroxylating 4-propylbenzoic acid (PBA) with NADPH-dependent cytochrome P450 oxidoreductase (CPR) as the single redox partner; the reaction efficiency was improved by the addition of electron transfer protein cytochrome b5 (Cyt-b5). Furthermore, CYP5150A2 exhibited substantial activity with redox partners Cyt-b5 and NADH-dependent Cyt-b5 reductase (CB5R) even in the absence of CPR. These results indicated that a combination of CB5R and Cyt-b5 may be capable of donating both the first and the second electrons required for the monooxygenation reaction. Under reaction conditions in which the redox system was associated with the CB5R-dependent Cyt-b5 reduction system, the exogenous addition of CPR and NADPH had no effect on the PBA hydroxylation rate or on coupling efficiency, indicating that the transfer of the second electron from Cyt-b5 was the rate-limiting step in the monooxygenase system. In addition, the rate of PBA hydroxylation was significantly dependent on Cyt-b5 concentration, exhibiting Michaelis-Menten kinetics. This study provides indubitable evidence that the combination of CB5R and Cyt-b5 is an alternative redox partner facilitating the monooxygenase reaction catalyzed by CYP5150A2.

Molecular identification and functional characterization of cytochrome P450 monooxygenases from the brown-rot basidiomycete Postia placenta.

We explored the molecular diversity and functional capabilities of cytochrome P450 monooxygenases (P450s) from the brown-rot basidiomycete Postia placenta. Using bioinformatic and experimental data, we found 250 genes of P450s in the whole genome, including 60 putative allelic variants. Phylogenetic analysis revealed the presence of 42 families, including 18 novel families. Comparative phylogenetic analysis of P450s from P. placenta and the white-rot basidiomycete Phanerochaete chrysosporium suggested that vigorous gene duplication and molecular evolution occurred after speciation of basidiomycetes. Among the 250 gene models, 184 were isolated as full-length cDNA and transformed into Saccharomyces cerevisiae to construct a functional library in which recombinant P450s were co-expressed with yeast NADPH-P450 oxidoreductase. Using this library, the catalytic potentials of P450s against a wide variety of compounds were investigated. A functionomic survey allowed the discovery of novel catalytic properties of P. placenta P450s. The phylogenetic diversity of the CYP53 family in P. placenta was clear, and CYP53D2 is capable of converting stilbene derivatives. This is the first report of this peculiar function of the CYP53 family. Our increased understanding of the molecular and functional diversity of P450s in this fungus will facilitate comprehension of metabolic diversity in basidiomycetes and has future biotechnology applications.

Molecular and functional diversity of fungal cytochrome P450s.

A series of genome projects have uncovered an astonishing molecular diversity of cytochrome P450s in the fungal kingdom. Fungal P450s discovered from such projects are often categorized into novel families and subfamilies. It thus appears that the divergence of fungal P450s is greater than of animal, plant, or bacterial P450s. The tremendous variation implies that fungi have vigorously diversified P450 functions to meet novel metabolic needs. To better understand the metabolic diversity of fungi, one must undertake the challenging task of exploiting the catalytic functions of numerous P450s. A compilation of P450 functions will also enable us to utilize their catalytic potentials in biotechnology. Experimental screening remains essential however, to elucidate the catalytic potentials of individual P450s. This review outlines the molecular and genomic aspects of fungal P450s, and introduces new functions revealed by functionomic studies using a recently developed, rapid, functional screening system.

Flavin-containing monooxygenases from Phanerochaete chrysosporium responsible for fungal metabolism of phenolic compounds.

We investigated the cellular responses of the white-rot basidiomycete Phanerochaete chrysosporium against vanillin. Based upon a proteomic survey, it was demonstrated that two flavin-containing monooxygenases (PcFMO1 and PcFMO2) are translationally up-regulated in response to exogenous addition of vanillin. To elucidate their catalytic functions, we cloned cDNAs and heterologously expressed them in Escherichia coli. The recombinant PcFMO1 showed catalytic activities against monocyclic phenols such as phenol, hydroquinone, and 4-chlorophenol. In addition, the product from hydroquinone was identified as 1,2,4-trihydroxybenzene, an important intermediate in a metabolic pathway of aromatic compounds in which the aromatic ring of 1,2,4-trihydroxybenzene can be further cleaved by fungal dioxygenases for mineralization. Thus, the ortho-cleavage pathway of phenolic compounds would presumably be associated with PcFMO1.

Latent Functions and Applications of Cytochrome P450 Monooxygenases from Thamnidium elegans: A Novel Biocatalyst for 14α-Hydroxylation of Testosterone.

Cytochrome P450 monooxygenases (P450s) are ubiquitous enzymes with high availability and diversity in nature. Fungi provide a diverse and complex array of P450s, and these enzymes play essential roles in various secondary metabolic processes. Besides the physiological impacts of P450s on fungal life, their versatile functions are attractive for use in advanced applications of the biotechnology sector. Herein, we report gene identification and functional characterization of P450s from the zygomycetous fungus Thamnidium elegans (TeCYPs). We identified 48 TeCYP genes, including two putative pseudogenes, from the whole-genome sequence of T. elegans. Furthermore, we constructed a functional library of TeCYPs and heterologously expressed 46 TeCYPs in Saccharomyces cerevisiae. Recombinants of S. cerevisiae were then used as whole-cell biocatalysts for bioconversion of various compounds. Catalytic potentials of various TeCYPs were demonstrated through a functionomic survey to convert a series of compounds, including steroidal substrates. Notably, CYP5312A4 was found to be highly active against testosterone. Based on nuclear magnetic resonance analysis, enzymatic conversion of testosterone to 14α-hydroxytestosterone by CYP5312A4 was demonstrated. This is the first report to identify a novel fungal P450 that catalyzes the 14α-hydroxylation of testosterone. In addition, we explored the latent potentials of TeCYPs using various substrates. This study provides a platform to further study the potential use of TeCYPs as catalysts in pharmaceutical and agricultural industries and biotechnology.

Latent potentials of the white-rot basidiomycete Phanerochaete chrysosporium responsible for sesquiterpene metabolism: CYP5158A1 and CYP5144C8 decorate (E)-α-bisabolene.

Basidiomycetes produce various sesquiterpenoids and their relevance for pharmaceutical and agricultural applications and understanding their biosynthetic machinery to produce these secondary metabolites have attracted significant interest. Because sesquiterpene synthases (STSs) and cytochrome P450 monooxygenases (P450s) play pivotal roles in the production of sesquiterpenoids, functional characterization of these enzymes is fundamentally essential. In this study, we found 11 possible STSs from the white-rot basidiomycete Phanerochaete chrysosporium (PcSTSs) and isolated nine of these as full-length cDNAs encoding a mature open reading frame. Using the isolated cDNAs, we performed heterologous expression of PcSTSs in Saccharomyces cerevisiae. Metabolic studies revealed that seven of the PcSTSs produce a series of sesquiterpene scaffolds, including (E)-α-bisabolene. Furthermore, we constructed a co-expression system of (E)-α-bisabolene synthase and P450 from P. chrysosporium (PcCYP). Semi-comprehensive screening using 120 isoforms of PcCYPs resulted in the identification of CYP5158A1 and CYP5144C8, two P450s capable of decorating (E)-α-bisabolene.

Insight into functional diversity of cytochrome P450 in the white-rot basidiomycete Phanerochaete chrysosporium: involvement of versatile monooxygenase.

To elucidate functional diversity of cytochrome P450 monooxygenases from the white-rot basidiomycete Phanerochaete chrysosporium (PcCYPs), we conducted a comprehensive functional screening using a wide variety of compounds. A functionomic survey resulted in characterization of novel PcCYP functions and discovery of versatile PcCYPs that exhibit broad substrate profiles. These results suggested that multifunctional properties of the versatile PcCYPs would play crucial roles in diversification of fungal metabolic systems involved in xenobiotic detoxification. To our knowledge, this is the first report describing multifunctional properties of versatile P450s from the fungal kingdom. An increased compilation of PcCYP functions will facilitate a thorough understanding of metabolic diversity in basidiomycetes and provide new insights that could also expedite practical applications in the biotechnology sector.

Construction and application of a functional library of cytochrome P450 monooxygenases from the filamentous fungus Aspergillus oryzae.

A functional library of cytochrome P450 monooxygenases from Aspergillus oryzae (AoCYPs) was constructed in which 121 isoforms were coexpressed with yeast NADPH-cytochrome P450 oxidoreductase in Saccharomyces cerevisiae. Using this functional library, novel catalytic functions of AoCYPs, such as catalytic potentials of CYP57B3 against genistein, were elucidated for the first time. Comprehensive functional screening promises rapid characterization of catalytic potentials and utility of AoCYPs.

Determination of a catalytic tyrosine in Trametes cervina lignin peroxidase with chemical modification techniques.

Trametes cervina lignin peroxidase (LiP) lacks a catalytic tryptophan strictly conserved in other LiP and versatile peroxidases. It contains tyrosine(181) at the potential catalytic site. This protein and the well-characterized Phanerochaete chrysosporium LiP with the catalytic tryptophan(171) have been chemically modified: the tryptophan-specific modification with N-bromosuccinimide sufficiently disrupted oxidation of veratryl alcohol by P. chrysosporium LiP, whereas the activity of T. cervina LiP was not affected, suggesting no catalytic tryptophan in T. cervina LiP. On the other hand, the tyrosine-specific modification with tetranitromethane did not affect the activities of P. chrysosporium LiP lacking tyrosine but inactivated T. cervina LiP due to the nitration of tyrosine(181). These results strongly suggest that tyrosine(181) is at the catalytic site in T. cervina LiP.

White-rot fungus Phanerochaete chrysosporium metabolizes chloropyridinyl-type neonicotinoid insecticides by an N-dealkylation reaction catalyzed by two cytochrome P450s.

We previously identified a cytochrome P450 (CYP) derived from the white-rot fungus Phanerochaete chrysosporium as involved in degradation of acetamiprid, a neonicotinoid (NEO) insecticide. In the present study, we investigated biodegradation of other NEOs by P. chrysosporium, and attempted to identify the CYP enzyme responsible for NEO degradation. P. chrysosporium was able to degrade some NEOs (acetamiprid, clothianidin, imidacloprid, and thiacloprid) in nutrient-rich medium. Two CYPs in P. chrysosporium (PcCYPs), CYP5037B3 and CYP5147A3, were identified as major isozymes involved in metabolism of three neonicotinoids that have in common a chloropyridinyl moiety (acetamiprid, imidacloprid, and thiacloprid) by screening yeast that heterologously express PcCYPs. Both PcCYPs catalyzed cleavage of the chloropyridinyl moiety and side chain of the three NEOs by N-dealkylation, resulting in 6-chloro-3-pyridinemethanol and respective side chain fragments. In a culture of P. chrysosporium, 97 % and 74 % of imidacloprid and thiacloprid were modified to form degradation products, and one of these, 6-chloro-3-pyridinemethanol, was further degraded. These two PcCYPs catalyzed almost the same reaction but their substrate specificity and expression pattern are slightly different. Altogether, we found that P. chrysosporium degrades NEOs via the activity of at least two different CYP isozymes.