Identification of Absidia orchidis steroid 11ß-hydroxylation system and its application in engineering Saccharomyces cerevisiae for one-step biotransformation to produce hydrocortisone.

Hydrocortisone is an effective anti-inflammatory drug and also an important intermediate for synthesis of other steroid drugs. The filamentous fungus Absidia orchidis is renowned for biotransformation of acetylated cortexolone through 11ß-hydroxylation to produce hydrocortisone. However, due to the presence of 11α-hydroxylase in A. orchidis, the 11α-OH by-product epi-hydrocortisone is always produced in a 1:1 M ratio with hydrocortisone. In order to decrease epi-hydrocortisone production, Saccharomyces cerevisiae was engineered in this work as an alternative way to produce hydrocortisone through biotransformation. Through transcriptomic analysis coupled with genetic verification in S. cerevisiae, the A. orchidis steroid 11ß-hydroxylation system was characterized, including a cytochrome P450 enzyme CYP5311B2 and its associated redox partners cytochrome P450 reductase and cytochrome b5. CYP5311B2 produces a mix of stereoisomers containing 11ß- and 11α-hydroxylation derivatives in a 4:1 M ratio. This fungal steroid 11ß-hydroxylation system was reconstituted in S. cerevisiae for hydrocortisone production, resulting in a productivity of 22 mg/L·d. Protein engineering of CYP5311B2 generated a R126D/Y398F variant, which had 3 times higher hydrocortisone productivity compared to the wild type. Elimination of C20-hydroxylation by-products and optimization of the expression of A. orchidis 11ß-hydroxylation system genes further increased hydrocortisone productivity by 238% to 223 mg/L·d. In addition, a novel steroid transporter ClCDR4 gene was identified from Cochliobolus lunatus, overexpression of which further increased hydrocortisone productivity to 268 mg/L·d in S. cerevisiae. Through increasing cell mass, 1060 mg/L hydrocortisone was obtained in 48 h and the highest productivity reached 667 mg/L·d. This is the highest hydrocortisone titer reported for yeast biotransformation system so far.

Absidia panacisoli sp. nov., isolated from rhizosphere of Panax notoginseng.

A strain (SYPF 7183T) was isolated from rhizosphere soil of Panax notoginseng in southwest China. Phylogenetic analyses indicated that strain SYPF 7183T was distinct from the other Absidia species with well-supported values. Strain SYPF 7183T produced spherical or subpyriform sporangia and short cylindrical sporangiospores. The azygospores were globose to oval. Based on morphological and phylogenetic evidence, the novel strain Absidia panacisoli sp. nov. is proposed.

The fate of mitochondria after infection of the Mucoralean fungus Absidia glauca by the fusion parasite Parasitella parasitica: comparison of mitochondrial genomes in zygomycetes.

Absidia glauca and Parasitella parasitica constitute a versatile experimental system for studying horizontal gene transfer between a mucoralean host and its fusion parasite. The A. glauca chondriome has a length of approximately 63 kb and a GC content of 28%. The chondriome of P. parasitica is larger, 83 kb, and contains 31% GC base pairs. These mtDNAs contain the standard fungal mitochondrial gene set, small and large subunit rRNAs, plus ribonuclease P RNA. Comparing zygomycete chondriomes reveals an unusually high number of homing endonuclease genes in P. parasitica, substantiating the mobility of intron elements independent of host-parasite interactions.

Two Novel Fungal Phenolic UDP Glycosyltransferases from Absidia coerulea and Rhizopus japonicus.

In the present study, two novel phenolic UDP glycosyltransferases (P-UGTs), UGT58A1 and UGT59A1, which can transfer sugar moieties from active donors to phenolic acceptors to generate corresponding glycosides, were identified in the fungal kingdom. UGT58A1 (from Absidia coerulea) and UGT59A1 (from Rhizopus japonicas) share a low degree of homology with known UGTs from animals, plants, bacteria, and viruses. These two P-UGTs are membrane-bound proteins with an N-terminal signal peptide and a transmembrane domain at the C terminus. Recombinant UGT58A1 and UGT59A1 are able to regioselectively and stereoselectively glycosylate a variety of phenolic aglycones to generate the corresponding glycosides. Phylogenetic analysis revealed the novelty of UGT58A1 and UGT59A1 in primary sequences in that they are distantly related to other UGTs and form a totally new evolutionary branch. Moreover, UGT58A1 and UGT59A1 represent the first members of the UGT58 and UGT59 families, respectively. Homology modeling and mutational analysis implied the sugar donor binding sites and key catalytic sites, which provided insights into the catalytic mechanism of UGT58A1. These results not only provide an efficient enzymatic tool for the synthesis of bioactive glycosides but also create a starting point for the identification of P-UGTs from fungi at the molecular level.IMPORTANCE Thus far, there have been many reports on the glycosylation of phenolics by fungal cells. However, no P-UGTs have ever been identified in fungi. Our study identified fungal P-UGTs at the molecular level and confirmed the existence of the UGT58 and UGT59 families. The novel sequence information on UGT58A1 and UGT59A1 shed light on the exciting and new P-UGTs hiding in the fungal kingdom, which would lead to the characterization of novel P-UGTs from fungi. Molecular identification of fungal P-UGTs not only is theoretically significant for a better understanding of the evolution of UGT families but also can be applied as a powerful tool in the glycodiversification of bioactive natural products for drug discovery.

Invasive zygomycosis in patients with graft-versus-host disease after allogeneic stem cell transplantation.

Invasive mold infections are a threat to immunosuppressed patients such as patients with graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). Up to 10% of SCT recipients develop invasive aspergillosis (IA). Invasive zygomycosis (IZ) may occur during treatment against IA. Here we report 4 SCT patients with GVHD diagnosed with IZ. All patients had received myeloablative hematopoietic SCT and developed chronic GVHD requiring systemic immunosuppression. Underlying diseases were acute lymphocytic leukemia (2), osteomyelofibrosis, and multiple myeloma. All patients had developed pulmonary infiltration that led to initiation of antifungal therapy. Treatment for IA was voriconazole, caspofungin, or itraconazole. Organs involved with zygomycosis were lung, nasal sinus, skin, and kidney. Treatment with liposomal amphotericin and posaconazole was initiated in all patients, and 2 patients also had surgical debridement as well. Despite intensive treatment, no patient survived. IZ is becoming more common in patients with GVHD on successful treatment for IA. Even non-specific symptoms are suspicious in this group of patients and need to be evaluated by vigorous diagnostics. Despite effective antifungals and surgical intervention, the prognosis is grim in patients with active GVHD, as immunoreconstitution is mandatory for successful management.

Molecular and phenotypic evaluation of Lichtheimia corymbifera (formerly Absidia corymbifera) complex isolates associated with human mucormycosis: rehabilitation of L. ramosa.

Thirty-eight isolates (including 28 isolates from patients) morphologically identified as Lichtheimia corymbifera (formerly Absidia corymbifera) were studied by sequence analysis (analysis of the internal transcribed spacer [ITS] region of the ribosomal DNA, the D1-D2 region of 28S, and a portion of the elongation factor 1alpha [EF-1alpha] gene). Phenotypic characteristics, including morphology, antifungal susceptibility, and carbohydrate assimilation, were also determined. Analysis of the three loci uncovered two well-delimited clades. The maximum sequence similarity values between isolates from both clades were 66, 95, and 93% for the ITS, 28S, and EF-1alpha loci, respectively, with differences in the lengths of the ITS sequences being detected (763 to 770 bp for isolates of clade 1 versus 841 to 865 bp for isolates of clade 2). Morphologically, the shapes and the sizes of the sporangiospores were significantly different among the isolates from both clades. On the basis of the molecular and morphological data, we considered isolates of clade 2 to belong to a different species named Lichtheimia ramosa because reference strains CBS 269.65 and CBS 270.65 (which initially belonged to Absidia ramosa) clustered within this clade. As neotype A. corymbifera strain CBS 429.75 belongs to clade 1, the name L. corymbifera was conserved for clade 1 isolates. Of note, the amphotericin B MICs were significantly lower for L. ramosa than for L. corymbifera (P < 0.005) but were always

Absidia parricida plays a dominant role in biotrophic fusion parasitism among mucoralean fungi (Zygomycetes): Lentamyces, a new genus for A. parricida and A. zychae.

Within the order Mucorales (Zygomycetes), the facultative parasites Parasitella parasitica, Chaetocladium brefeldii, Chaetocladium jonesii and Absidia parricida are known to initiate biotrophic fusion parasitic interactions on a wide variety of other mucoralean hosts. Their phylogenetic relationship within the Mucorales and their ability to form parasitic structures with several known host species was examined. Together with interspecific reactions between the mycoparasites, this study found: (i) no evident differences in the spectrum of non-parasitic hosts tested within the study; (ii) A. parricida parasitises all other fusion parasites; (iii) A. parricida is ancestral to all other parasites; (iv) A. parricida is reported for the first time as phylogenetically basal to all other mucoralean fungi except the Umbelopsidaceae and (v) based on phylogenetic analyses and physiological and morphological characteristics, the slow-growing species A. parricida and Absidia zychae are removed from the genus Absidia and reclassified in the newly described genus Lentamyces.

Primary cutaneous zygomycosis due to Absidia corymbifera in a patient with cutaneous T cell lymphoma.

A slowly enlarging arm ulcer appeared in a 61-year-old man with cutaneous T cell lymphoma. Skin biopsy revealed aseptate hyphae and nodular small/medium-sized pleomorphic CD4(+) T cell infiltration. Cultures yielded Absidia corymbifera which was identified by phenotypic and molecular methods. Since a thorough examination did not detect organ involvement, the patient was diagnosed as having primary cutaneous zygomycosis. This is the first case report of cutaneous zygomycosis caused by A. corymbifera in a patient with primary cutaneous CD4(+) small/medium-sized pleomorphic T-cell lymphoma. Other cases of primary cutaneous zygomycosis caused by A. corymbifera are also reviewed.

Revision of the genus Absidia (Mucorales, Zygomycetes) based on physiological, phylogenetic, and morphological characters; thermotolerant Absidia spp. form a coherent group, Mycocladiaceae fam. nov.

The genus Absidia comprises ubiquitously distributed soil fungi inhabiting different growth temperature optima ranging from 20-42 degrees C. Some of the mesophilic species are important biotechnologically in the biotransformation of steroids or as producers of rennin-like components, whereas species with higher growth temperature optima are of clinical relevance as opportunistic human pathogens. The aim of this study was to investigate the phylogenetic relationships between these species and to establish a revision of their systematics. For this purpose single and combined genealogies based on distance, MP, ML, and Bayesian analyses of aligned nucleotide sequences of the nuclear-encoded genes for actin (act) and for the 5.8S ribosomal RNA flanked by the ITS regions 1 and 2 (comprising 807 and 828 characters, respectively) of 16 Absidia species were reconstructed. The phylogenetic reconstructions suggest a trichotomy of the Absidia genus consisting of a mesophilic, a fast-growing thermotolerant, and a slowly-growing mycoparasitic Absidia group. The trichotomous phylogenetic grouping is concordant with the morphology of the zygospores, which are zygotes resulting from sexual conjugation between two compatible mating partners. Whereas the mesophilic group comprises the majority of absidiaceaeous species forming sterile hair-like, mycelial appendages on the suspensors of their zygospores, the thermotolerant group is characterised by the formation of smooth-walled zygospores, and the mycoparasitic group, namely Absidia parricida and A. zychae, by Mucor-like rough-walled zygospores. Based on the phylogenetic coherence of mesophilic and thermotolerant Absidia species, we propose that the two groups are separated into two distinct genera, Absidia for the mesophilic Absidia species resembling the Absidiaceae and Mycocladus for the thermotolerant species A. corymbifera, A. blakesleeana and A. hyalospora. Because Mycocladus is physiologically, phylogenetically, and morphologically distinct from the Absidiaceae s. str. we suggest that they are classified as a separate family, Mycocladiaceae fam. nov., which comprises the three species M. corymbifer, M. blakesleeanus and M. hyalospora.

Lipids of the zygomycete Absidia corymbifera F-965.

The cell lipids of the zygomycete Absidia corymbifera F-965 extracted with isopropanol and CHCl3-MeOH mixtures at the exponential growth phase comprise 20+/-2% of mycelium dry wt. The lipids consist of: triacylglycerols (51% of the total lipids extracted), diacylglycerols (9%), monoacylglycerols (3%), ergosterol (5%), ergosterol peroxide (5alpha,8alpha-epidioxyergosta-6,22-diene-3beta-ol) (3%), fatty-acid esters of ergosterol (less than 0.5%), free fatty acids (4%), glucocerebroside (3%), and glycerophospholipids (22%). The main phospholipids are phosphatidylethanolamine (39% of the total phospholipids), phosphatidyl-myo-inositol (17%), diphosphatidylglycerol (12%), phosphatidic acid (7%), phosphatidylcholine (6%), phosphatidylglycerol (3%), and two unusual phospholipids reported earlier, N-acetylphosphatidylethanolamine (7%) and N-ethoxycarbonyl phosphatidylethanolamine (9%). In addition, two unknown acidic phospholipids are present in traces. Saturated fatty acids of the lipids are dominated by n-hexadecanoic acid and unsaturated ones by octadecenoic acid; octadecadienoic and octadecatrienoic acids are present in lesser amounts. Ergosterol peroxide as well as the above glucocerebroside which contains 9-methylsphinga-4(E),9(E)-dienine have first been revealed in zygomycetes.

Two unusual glycerophospholipids from a filamentous fungus, Absidia corymbifera.

The chloroform-methanol extractable lipids of the soil filamentous fungus Absidia corymbifera VKMF-965 account for about 20% by weight of dry cells and are composed of low-polarity constituents (about 75% of the total lipids), such as triacylglycerols (mainly), diacylglycerols, sterols and free fatty acids, as well as of glycolipids (about 3%) and phospholipids. The last consist largely of components common to the fungal lipids, namely, phosphatidylethanolamine (38% of the total phospholipids), phosphatidyl-myo-inositol (16%), diphosphatidylglycerol (12%), phosphatidylcholine (7%), phosphatidic acid (6%) and phosphatidylglycerol (3%), and two unusual phospholipids, PL1 (6%) and PL2 (9%). Based on the infrared (IR), (1)H-nuclear magnetic resonance (NMR), (13)C-NMR and mass spectra along with the results of degradation experiment, these two phospholipids have been established to be 1,2-diacyl-sn-glycero-3-phospho(N-acetylethanolamine), or N-acetyl phosphatidylethanolamine, and 1,2-diacyl-sn-glycero-3-phospho(N-ethoxycarbonyl-ethanolamine), respectively. These structures have been confirmed by preparing similar phospholipids from the phosphatidylethanolamine isolated from the same fungus and correlating their chromatographic behaviour, IR and (1)H-NMR spectra with those of PL1 and PL2. So far N-acetyl phosphatidylethanolamine has been detected only in cattle and human brains and a human placenta but its structure was not rigorously proved. PL2 is a novel lipid; to our knowledge no natural phospholipid with an urethane group has yet been found. The main fatty acids of both the phospholipids are n-hexadecanoic, octadecanoic and octadecadienoic ones; PL2 contains in addition a considerable amount of octadecatrienoic acid with its greater portion located at the sn-1 position.

Green fluorescent protein as a reporter for gene expression in the mucoralean fungus Absidia glauca.

Mucoralean fungi (Zygomycota) are used for many industrial processes and also as important model organisms for investigating basic biological problems. Their genetic analysis is severely hampered by low transformation frequencies, by their strong tendency towards autonomous replication of plasmids instead of stable integration, and by the lack of reliable genetic reporter systems. We constructed plasmids for transforming the model zygomycete Absidia glauca that carry the versatile reporter gene coding for green fluorescent protein (GFP). gfp expression is controlled either by the homologous actin promoter or the promoter for the elongation factor of translation, EF1alpha. These plasmids also confer neomycin resistance and carry one of two genetic elements (rag1, seg1) that improve mitotic stability of the plasmid. The gfp constructs were replicated extrachromosomally and could be recovered from retransformed Escherichia coli cells. gfp expression was monitored by epifluorescence microscopy. The gfp reporter gene plasmids presented here for the model zygomycete A. glauca constitute the first reliable system that allows the monitoring of gene expression in this important group of fungi.

Reliable amplification of actin genes facilitates deep-level phylogeny.

The gene for actin as a highly conserved and functionally essential genetic element is developing into a major tool for phylogenetic analysis within a broad organismic range. We therefore propose a set of universally applicable primers that allow reliable amplification of actin genes. For primer construction the amino acid sequences of 57 actin genes comprising fungi, animals, plants and protists were analysed, aligned and used for the definition of six well-conserved regions which are suitable as priming sites in PCR amplification experiments. Ten primers were designed for specific in vitro amplification of actin gene fragments from a wide range of microorganisms. The corresponding gene fragments provide a strong basis to isolate nearly complete actin genes for further molecular characterization and for establishing phylogenies based on actin gene trees.