Cloning, expression, and characterization of Baeyer-Villiger monooxygenases from eukaryotic Exophiala jeanselmei strain KUFI-6N.

The fungus Exophiala jeanselmei strain KUFI-6N produces a unique cycloalkanone monooxygenase (ExCAMO) that displays an uncommon substrate spectrum of Baeyer-Villiger oxidation of 4-10-membered ring ketones. In this study, we aimed to identify and sequence the gene encoding ExCAMO from KUFI-6N and overexpress the gene in Escherichia coli. We found that the primary structure of ExCAMO is most closely related to the cycloalkanone monooxygenase from Cylindrocarpon radicicola ATCC 11011, with 54.2% amino acid identity. ExCAMO was functionally expressed in E. coli and its substrate spectrum and kinetic parameters were investigated. Substrate profiling indicated that ExCAMO is unusual among known Baeyer-Villiger monooxygenases owing to its ability to accept a variety of substrates, including C4-C12 membered ring ketones. ExCAMO has high affinity and catalytic efficiency toward cycloalkanones, the highest being toward cyclohexanone. Five other genes encoding Baeyer-Villiger monooxygenases were also cloned and expressed in E. coli.

Expanding the Toolbox of R-Selective Amine Transaminases by Identification and Characterization of New Members.

Amine transaminases (ATAs) are used to synthesize enantiomerically pure amines, which are building blocks for pharmaceuticals and agrochemicals. R-selective ATAs belong to the fold type IV PLP-dependent enzymes, and different sequence-, structure- and substrate scope-based features have been identified in the past decade. However, our knowledge is still restricted due to the limited number of characterized (R)-ATAs, with additional bias towards fungal origin. We aimed to expand the toolbox of (R)-ATAs and contribute to the understanding of this enzyme subfamily. We identified and characterized four new (R)-ATAs. The ATA from Exophiala sideris contains a motif characteristic for d-ATAs, which was previously believed to be a disqualifying factor for (R)-ATA activity. The crystal structure of the ATA from Shinella is the first from a Gram-negative bacterium. The ATAs from Pseudonocardia acaciae and Tetrasphaera japonica are the first characterized (R)-ATAs with a shortened/missing N-terminal helix. The active-site charges vary significantly between the new and known ATAs, correlating with their diverging substrate scope.

Hydroxylation of steroids by Fusarium oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa.

The potential of Fusarium oxysporum var. cubense UAMH 9013 to perform steroid biotransformations was reinvestigated using single phase and pulse feed conditions. The following natural steroids served as substrates: dehydroepiandrosterone (1), pregnenolone (2), testosterone (3), progesterone (4), cortisone (5), prednisone (6), estrone (7) and sarsasapogenin (8). The results showed the possible presence of C-7 and C-15 hydroxylase enzymes. This hypothesis was explored using three synthetic androstanes: androstane-3,17-dione (9), androsta-4,6-diene-3,17-dione (10) and 3α,5α-cycloandrost-6-en-17-one (11). These fermentations of non-natural steroids showed that C-7 hydroxylation was as a result of that position being allylic. The evidence also pointed towards the presence of a C-15 hydroxylase enzyme. The eleven steroids were also fed to Exophialajeanselmei var. lecanii-corni UAMH 8783. The results showed that the fungus appears to have very active 5α and 14α-hydroxylase enzymes, and is also capable of carrying out allylic oxidations. Ceratocystis paradoxa UAMH 8784 was grown in the presence of the above-mentioned steroids. The results showed that monooxygenases which effect allylic hydroxylation and Baeyer-Villiger rearrangement were active. However, redox reactions predominated.

RNA interference of WdFKS1 mRNA expression causes slowed growth, incomplete septation and loss of cell wall integrity in yeast cells of the polymorphic, pathogenic fungus Wangiella (Exophiala) dermatitidis.

As one of the main components of the fungal cell wall, ß-1,3-glucan provides the mechanical strength to protect fungal protoplasts. The enzyme responsible for the synthesis of ß-1,3-glucans in fungi is ß-1,3-glucan synthase. Here we report the cloning, sequencing and characterization of the WdFKS1 gene, which in the pathogenic fungus Wangiella dermatitidis encodes the catalytic domain of its ß-1, 3-glucan synthase. Because our research suggested that WdFKS1 is a single copy essential gene, we used RNA interference to reduce its expression. Reduction of the WdFKS1 messenger retarded growth and caused the loss of cell wall integrity of yeast cells, but not hyphae or sclerotic cells. We suggest that the WdFKS1 in this polymorphic agent of phaeohyphomycosis is not only required for cell wall construction and maintenance, but also is involved in septum formation.

Immunoaffinity purification of the class V chitin synthase of Wangiella (Exophiala) dermatitidis.

The class V chitin synthase is unique because it has a myosin motor-like domain fused to its catalytic domain. The biochemical properties of this enzyme and its function remain undefined beyond the knowledge that it is the only single chitin synthase required for sustained cell growth at elevated temperatures and, consequently, virulence. This report describes our successful efforts to isolate and purify an active and soluble form of the enzyme from the cell membranes of Wangiella by using a specific polyclonal antibody. To our knowledge, this is the first purification of a single chitin synthase of a filamentous fungus.

Cytolocalization of the class V chitin synthase in the yeast, hyphal and sclerotic morphotypes of Wangiella (Exophiala) dermatitidis.

Wangiella (Exophiala) dermatitidis is a polymorphic fungus that produces polarized yeast and hyphae, as well as a number of non-polarized sclerotic morphotypes. The phenotypic malleability of this agent of human phaeohyphomycosis allows detailed study of its biology, virulence and the regulatory mechanisms responsible for the transitions among the morphotypes. Our prior studies have demonstrated the existence of seven chitin synthase structural genes in W. dermatitidis, each of which encodes an isoenzyme of a different class. Among them, the class V chitin synthase (WdChs5p) is most unique in terms of protein structure, because it has an N-terminal myosin motor-like domain with a P-loop (MMD) fused to its C-terminal chitin synthase catalytic domain (CSCD). However, the exact role played by WdChs5p in the different morphotypes remains undefined beyond the knowledge that it is the only single chitin synthase required for sustained cell growth at 37 degrees C and consequently virulence. This report describes the expression in Escherichia coli of a 12kDa polypeptide (WdMyo12p) of WdChs5p, which was used to raise in rabbits a polyclonal antibody that recognized exclusively its MMD region. Results from the use of the antibody in immunocytolocalization studies supported our previous findings that WdChs5p is critically important at infection temperatures for maintaining the cell wall integrity of developing yeast buds, elongating tips of hyphae, and random sites of expansion in sclerotic forms. The results also suggested that WdChs5p localizes to the regions of cell wall growth in an actin-dependent fashion.

Transcription and expression analyses of WdCHS5, which encodes a class V chitin synthase with a myosin motor-like domain in Wangiella (Exophiala) dermatitidis.

WdChs5p in Wangiella dermatitidis is a class V chitin synthase that is required for sustained cell growth at the temperature of infection (37 degrees C) and its encoding gene, WdCHS5, has a differential expression feature. Nuclear run-on and mRNA stability assays showed that increased WdCHS5 mRNA synthesis was the major factor responsible for the increased WdCHS5 transcript at 37 degrees C. Epitope tagging of WdChs5p in W. dermatitidis showed that the WdChs5p-myc protein had a differential expression feature that was similar to the differential transcription of the WdCHS5 gene. In conclusion, it is shown that transcriptional regulation is the first and probably the most important control point of the expression of WdCHS5.

Effects of disrupting the polyketide synthase gene WdPKS1 in Wangiella [Exophiala] dermatitidis on melanin production and resistance to killing by antifungal compounds, enzymatic degradation, and extremes in temperature.

BACKGROUND: Wangiella dermatitidis is a human pathogenic fungus that is an etiologic agent of phaeohyphomycosis. W. dermatitidis produces a black pigment that has been identified as a dihydroxynaphthalene melanin and the production of this pigment is associated with its virulence. Cell wall pigmentation in W. dermatitidis depends on the WdPKS1 gene, which encodes a polyketide synthase required for generating the key precursor for dihydroxynaphthalene melanin biosynthesis. RESULTS: We analyzed the effects of disrupting WdPKS1 on dihydroxynaphthalene melanin production and resistance to antifungal compounds. Transmission electron microscopy revealed that wdpks1Delta-1 yeast had thinner cell walls that lacked an electron-opaque layer compared to wild-type cells. However, digestion of the wdpks1Delta-1 yeast revealed small black particles that were consistent with a melanin-like compound, because they were acid-resistant, reacted with melanin-binding antibody, and demonstrated a free radical signature by electron spin resonance analysis. Despite lacking the WdPKS1 gene, the mutant yeast were capable of catalyzing the formation of melanin from L-3,4-dihyroxyphenylalanine. The wdpks1Delta-1 cells were significantly more susceptible to killing by voriconazole, amphotericin B, NP-1 [a microbicidal peptide], heat and cold, and lysing enzymes than the heavily melanized parental or complemented strains. CONCLUSION: In summary, W. dermatitidis makes WdPKS-dependent and -independent melanins, and the WdPKS1-dependent deposition of melanin in the cell wall confers protection against antifungal agents and environmental stresses. The biological role of the WdPKS-independent melanin remains unclear.

Quantification of homogentisate-1,2-dioxygenase expression in a fungus degrading ethylbenzene.

A quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) assay was utilized to quantify the expression of ElHDO in the fungus Exophiala lecanii-corni during the biodegradation of ethylbenzene and other volatile organic pollutants. The assay was applied to measure the impact of pollutant mixtures on ElHDO expression relative to that of a housekeeping gene (18S rRNA). Three compounds were tested in mixtures with ethylbenzene: methyl propyl ketone, phenylacetate and o-xylene. These chemicals repressed, induced, or had no effect on ethylbenzene degradation, respectively. The results demonstrate that the gene target expression value (T(N)) is a useful parameter for evaluating the effect of pollutant mixtures on gene expression. T(N) was found to reflect macroscopic changes in ethylbenzene utilization rates although these two parameters were not related in a linear fashion for all compounds. The assay was log-linear over 5 orders of magnitude of RNA concentration and reproducible between samples (the largest T(N) standard deviation was 20%). The comparative qRT-PCR assay used in this research represents a viable alternative to absolute quantification methods to monitor in situ fungal gene expression in natural and engineered environmental systems.

WdChs1p, a class II chitin synthase, is more responsible than WdChs2p (Class I) for normal yeast reproductive growth in the polymorphic, pathogenic fungus Wangiella (Exophiala) dermatitidis.

The chitin synthase gene WdCHS1 was isolated from a partial genomic DNA library of the pathogenic polymorphic fungus Wangiella dermatitidis. Sequencing showed that WdCHS1 encoded a class II chitin synthase composed of 988 amino acids. Disruption of WdCHS1 produced strains that were hyperpigmented in rich media, grew as yeast at wild-type rates at both 25 and 37 degrees C and were as virulent as the wild type in a mouse model. However, detailed morphological and cytological studies of the wdchs1Delta mutants showed that yeast cells often failed to separate, tended to be enriched with chitin in septal regions and, sometimes, were enlarged with multiple nuclei, had broader mother cell-daughter bud regions and had other cell wall defects seen considerably less often than in the wild type or wdchs2 Delta strains. Disruption of WdCHS1 and WdCHS2 in the same background revealed that WdChs1p had functions synergistic to those of WdChs2p, because mutants devoid of both isozymes produced growth that was very abnormal at 25 degrees C and was not viable at 37 degrees C unless osmotically stabilized. These results suggested that WdChs1p was more responsible than WdChs2p for normal yeast cell reproductive growth because strains with defects in the latter exhibited no morphological abnormalities, whereas those with defects in WdChs1p were frequently impaired in one or more yeast developmental processes.

Characterization of two polyketide synthase genes in Exophiala lecanii-corni, a melanized fungus with bioremediation potential.

Exophiala lecanii-corni has significant bioremediation potential because it can degrade a wide range of volatile organic compounds. In order to identify sites for the insertion of genes that might enhance this potential, a genetic analysis of E. lecanii-corni was undertaken. Two polyketide synthase genes, ElPKS1 and ElPKS2, have now been discovered by a PCR-based strategy. ElPKS1 was isolated by a marker rescue technique. The nucleotide sequence of ElPKS1 consists of a 6576-bp open reading frame encoding a protein with 2192 amino acids, which was interrupted by a 60-bp intron near the 5' end and a 54-bp intron near the 3' end. Sequence analysis, results from disruption experiments, and physiological tests showed that ElPKS1 encoded a polyketide synthase required for melanin biosynthesis. Since ElPKS1 is non-essential, it is a desirable bioengineering target site for the insertion of native and foreign genes. The successful expression of these genes could enhance the bioremediation capability of the organism. ElPKS2 was cloned by colony hybridization screening of a partial genomic library with an ElPKS2 PCR product. ElPKS2 had a 6465-bp open reading frame that encoded 2155 amino acids and had introns of 56, 67, 54, and 71 bp. Although sequence analysis of the derived protein of ElPKS2 confirmed the polyketide synthase nature of its protein product, the function of that product remains unclear.

Wangiella (Exophiala) dermatitidis WdChs5p, a class V chitin synthase, is essential for sustained cell growth at temperature of infection.

The chitin synthase structural gene WdCHS5 was isolated from the black fungal pathogen of humans Wangiella dermatitidis. Sequence analysis revealed that the gene has a myosin motor-like-encoding region at its 5' end and a chitin synthase (class V)-encoding region at its 3' end. Northern blotting showed that WdCHS5 is expressed at high levels under conditions of stress. Analysis of the 5' upstream region of WdCHS5 fused to a reporter gene indicated that one or more of the potential regulatory elements present may have contributed to the high expression levels. Disruption of WdCHS5 produced mutants that grow normally at 25 degrees C but have severe growth and cellular abnormalities at 37 degrees C. Osmotic stabilizers, such as sorbitol and sucrose, rescued the wild-type phenotype, which indicated that the loss of WdChs5p causes cell wall integrity defects. Animal survival tests with a mouse model of acute infection showed that all wdchs5Delta mutants are less virulent than the parental strain. Reintroduction of the WdCHS5 gene into the wdchs5Delta mutants abolished the temperature-sensitive phenotype and reestablished their virulence. We conclude that the product of WdCHS5 is required for the sustained growth of W. dermatitidis at 37 degrees C and is of critical importance to its virulence.

Compensatory expression of five chitin synthase genes, a response to stress stimuli, in Wangiella (Exophiala) dermatitidis, a melanized fungal pathogen of humans.

Numerous chitin synthase structural (CHS) genes have been identified in fungi, and usually there are several CHS genes per species. Compensatory expression of one CHS gene in response to defects in other CHS genes has not been reported. Five chitin synthase structural (WdCHS) genes have been identified in the melanized human pathogen Wangiella dermatitidis: WdCHS1, WdCHS2, WdCHS3, WdCHS4 and WdCHS5. This study showed that increased WdCHS expression existed as a compensatory mechanism in response to stress induced by chitin synthase gene disruptions, or by exposure of the wild-type or two temperature-sensitive morphological mutants, for short or long periods, to 37 degrees C. In general, the compensatory responses varied with each WdCHS gene, and in accordance with the hypothesized functions of the chitin synthase (WdChsp) encoded. It is suggested that these compensatory responses indicate that WdCHS gene transcription in W. dermatitidis functions as part of a cell-wall integrity pathway in a manner similar to that recently described for Saccharomyces cerevisiae.

Characterization of WdChs3p, a class III chitin synthase, of Wangiella (Exophiala) dermatitidis, overexpressed in Saccharomyces cerevisiae.

The class III chitin synthase (WdChs3p) of the fungal pathogen of humans Wangiella dermatitidis is expressed differentially under stress growth conditions and, together with WdChs2p, it contributes to virulence. However, because this fungus produces four other chitin synthases, and a quadruple disruption strain with only WdChs3p activity has not been derived, we characterized this enzyme further after heterologous expression of the WdCHS3 cDNA under the regulation of the inducible GAL1 promoter in Saccharomyces cerevisiae. In contrast to our prior conclusions about the zymogenic nature of WdChs3p produced in W. dermatitidis, the enzyme expressed in yeast had a non-zymogenic activity, that is, an activity not potentiated by controlled proteolysis. Nonetheless, other characteristics of WdChs3p activity expressed in yeast were similar to those of the enzyme from its parent, including being stable over a broad range of pH and temperature values, which should facilitate attempts to purify this unique chitin synthase and better define its structure.

WdChs2p, a class I chitin synthase, together with WdChs3p (class III) contributes to virulence in Wangiella (Exophiala) dermatitidis.

The chitin synthase structural gene WdCHS2 was isolated by screening a subgenomic DNA library of Wangiella dermatitidis by using a 0.6-kb PCR product of the gene as a probe. The nucleotide sequence revealed a 2,784-bp open reading frame, which encoded 928 amino acids, with a 59-bp intron near its 5' end. Derived protein sequences showed highest amino acid identities with those derived from the CiCHS1 gene of Coccidioides immitis and the AnCHSC gene of Aspergillus nidulans. The derived sequence also indicated that WdChs2p is an orthologous enzyme of Chs1p of Saccharomyces cerevisiae, which defines the class I chitin synthases. Disruptions of WdCHS2 produced strains that showed no obvious morphological defects in yeast vegetative growth or in ability to carry out polymorphic transitions from yeast cells to hyphae or to isotropic forms. However, assays showed that membranes of wdchs2Delta mutants were drastically reduced in chitin synthase activity. Other assays of membranes from a wdchs1Deltawdchs3Deltawdchs4Delta triple mutant showed that their residual chitin synthase activity was extremely sensitive to trypsin activation and was responsible for the majority of zymogenic activity. Although no loss of virulence was detected when wdchs2Delta strains were tested in a mouse model of acute infection, wdchs2Deltawdchs3Delta disruptants were considerably less virulent in the same model, even though wdchs3Delta strains also had previously shown no loss of virulence. This virulence attenuation in the wdchs2Deltawdchs3Delta mutants was similarly documented in a limited fashion in more-sensitive cyclophosphamide-induced immunocompromised mice. The importance of WdChs2p and WdChs3p to the virulence of W. dermatitidis was then confirmed by reconstituting virulence in the double mutant by the reintroduction of either WdCHS2 or WdCHS3 into the wdchs2Deltawdchs3Delta mutant background.

Identification, classification, and phylogeny of the pathogenic species Exophiala jeanselmei and related species by mitochondrial cytochrome b gene analysis.

We analyzed a 402-bp sequence of the mitochondrial cytochrome b gene of 34 strains of Exophiala jeanselmei and 16 strains representing 12 related species. The strains of E. jeanselmei were classified into 20 DNA types and 17 amino acid types. The differences between these strains were found in 1 to 60 nucleotides and 1 to 17 amino acids. On the basis of the identities and similarities of nucleotide and amino acid sequences, some strains were reidentified: i.e., two strains of E. jeanselmei var. hetermorpha and one strain of E. castellanii as E. dermatitidis (including the type strain), three strains of E. jeanselmei as E. jeanselmei var. lecanii-corni (including the type strain), three strains of E. jeanselmei as E. bergeri (including the type strain), seven strains of E. jeanselmei as E. pisciphila (including the type strain), seven strains of E. jeanselmei as E. jeanselmei var. jeanselmei (including the type strain), one strain of E. jeanselmei as Fonsecaea pedrosoi (including the type strain), and one strain of E. jeanselmei as E. spinifera (including the type strain). Some E. jeanselmei strains showed distinct nucleotide and amino acid sequences. The amino-acid-based UPGMA (unweighted pair group method with the arithmetic mean) tree exhibited nearly the same topology as those of the DNA-based trees obtained by neighbor joining, maximum parsimony, and maximum likelihood methods.

WdCHS3, a gene that encodes a class III chitin synthase in Wangiella (Exophiala) dermatitidis, is expressed differentially under stress conditions.

Class III chitin synthases are important for hyphal growth in some filamentous fungi but are not found in yeasts. Using a specific PCR product that encodes a portion of the class III chitin synthase of W. dermatitidis as a probe, we isolated the chitin synthase gene, WdCHS3, from this polymorphic melanized pathogen of humans. Northern blotting showed that WdCHS3 was highly expressed under stress conditions, such as the shift of cells to temperatures commensurate with infection, or to conditions that induce cellular morphogenesis in this fungus. Analysis of the 5' upstream sequence of WdCHS3 provided evidence for a negative regulatory element at between -780 and -1600 bp. Western blotting indicated that the production of the WdChs3p was temperature dependent and temporally regulated. Disruption of WdCHS3 in a wild-type strain and in two temperature-sensitive morphological mutants resulted in significantly reduced chitin synthase activities but did not obviously affect their morphologies, growth rates, chitin contents, or virulence. This paradox suggested that the contributions of the high levels of WdCHS3 gene expression and WdChs3p production in strains subjected to stress reside in unknown or unexamined parts of the life cycle of this ecologically poorly known member of the Fungi Imperfecti. Nonetheless, this report presents the first evidence that transcription of a chitin synthase gene is regulated by a negative regulatory element in its 5' upstream sequence.

Purification and characterization of cyclohexanone 1,2-monooxygenase from Exophiala jeanselmei strain KUFI-6N.

Baeyer-Villiger cyclohexanone 1,2-monooxygenase (CHMO) was purified 17.1-fold from cell extracts of the fungus Exophiala jeanselmei grown on cyclohexanol to electrophoretically homogeneity by serial chromatographies. The molecular mass of the native enzyme was approximately 74 kDa by gel filtration and SDS-PAGE. Some enzymic characterizations were studied. The NH2-terminal amino acid residues were Ala-Lys-Ser-Leu-Asp-Val-Leu-Ile-Val-Gly-Ala-Gly-Phe-Gly-Gly-Ile-Tyr-Gln-Leu-, with similarity to the bacterial CHMOs of FAD-binding and NADPH-dependent type Baeyer-Villiger monooxygenases.

WdChs4p, a homolog of chitin synthase 3 in Saccharomyces cerevisiae, alone cannot support growth of Wangiella (Exophiala) dermatitidis at the temperature of infection.

By using improved transformation methods for Wangiella dermatitidis, and a cloned fragment of its chitin synthase 4 structural gene (WdCHS4) as a marking sequence, the full-length gene was rescued from the genome of this human pathogenic fungus. The encoded chitin synthase product (WdChs4p) showed high homology with Chs3p of Saccharomyces cerevisiae and other class IV chitin synthases, and Northern blotting showed that WdCHS4 was expressed at constitutive levels under all conditions tested. Reduced chitin content, abnormal yeast clumpiness and budding kinetics, and increased melanin secretion resulted from the disruption of WdCHS4 suggesting that WdChs4p influences cell wall structure, cellular reproduction, and melanin deposition, respectively. However, no significant loss of virulence was detected when the wdchs4Delta strain was tested in an acute mouse model. Using a wdchs1Delta wdchs2Delta wdchs3Delta triple mutant of W. dermatitidis, which grew poorly but adequately at 25 degrees C, we assayed WdChs4p activity in the absence of activities contributed by its three other WdChs proteins. Maximal activity required trypsin activation, suggesting a zymogenic nature. The activity also had a pH optimum of 7.5, was most stimulated by Mg(2+), and was more inhibited by polyoxin D than by nikkomycin Z. Although the WdChs4p activity had a broad temperature optimum between 30 to 45 degrees C in vitro, this activity alone did not support the growth of the wdchs1Delta wdchs2Delta wdchs3Delta triple mutant at 37 degrees C, a temperature commensurate with infection.