Molecular and Morphological Confirmation of Three Undescribed Species of Mortierella from Korea

Three fungal isolates designated as CNUFC-YR329-1, CNUFC-PTS103-1, and CNUFC-PTS2-1 were discovered during a survey of fungal diversity of the order Mortierellales from freshwater and pine tree rhizosphere soil samples in Korea. The strains were analyzed morphologically and phylogenetically based on the internal transcribed spacer (ITS) and large subunit (LSU) of ribosomal DNA gene sequences. Based on their morphology and phylogeny, the three isolates were identified as Mortierella elongata, M. horticola, and M. humilis, respectively. To the best of our knowledge, M. elongata, M. horticola, and M. humilis, belonging to an undiscovered taxon, have not been previously described in Korea.

Distribution of Rhizosphere and Endosphere Fungi on the First-Class Endangered Plant Cypripedium japonicum

Endangered native plant habitats and populations are rapidly disappearing because of climate and environmental changes. As a representative, the abundance of the first-class endangered wild plant, Cypripedium japonicum, has been rapidly decreasing in Korea. The purpose of this study was to evaluate the distribution of rhizosphere and endophytic fungi on C. japonicum in its native habitat. A total of 440 rhizosphere and 79 endosphere fungi isolates were isolated and identified on the basis of their molecular characteristics. Sixty-five genera and 119 fungi species were identified in this study. The genus Trichoderma showed the highest abundance among both rhizosphere and endosphere fungi. Mortierella, Hypocrea, and Penicillium spp. were also relatively dominant species on C. japonicum. The community structures of rhizosphere and endosphere fungi were similar, but endosphere fungi showed greater diversity.

Regio- and stereo-selective hydroxylations of ingenane diterpenoids by Mortierella ramanniana and Gibberella fujikuroi / 中国天然药物

The regio- and stereo-selective hydroxylations of two ingenane diterpenoids, 20-deoxyingenol (1) and 13-oxyingenol dodecanoat (2), by the filamentous fungi Mortierella ramanniana and Gibberella fujikuroi were investigated in the present study. Four undescribed metabolites (3-6) of substrate 1 and two undescribed metabolites (7 and 8) of substrate 2 were isolated. All the metabolites were identified as hydroxylated ingenane derivatives by extensive NMR and HR-ESI-MS data analyses. All the biotransformed compounds and the substrates were evaluated for their cytotoxicities against three human cancer cell lines, including human colon cancer Caco-2, breast cancer MCF-7, and adriamycin (ADM)-resistant MCF-7/ADM cell lines. All ingenane alcohols (1, and 3-6) displayed no significant cytotoxic activities. The substrate 13-oxyingenol dodecanoat (2) showed moderate cytotoxicity with IC values being 35.59 ± 5.37 μmol·L (Caco-2), 24.04 ± 4.70 μmol·L (MCF-7), and 22.24 ± 5.19 μmol·L (MCF-7/ADM). However, metabolites 7 and 8 displayed no significant cytotoxicity. These results indicated that the hydroxylation at the C-13 aliphatic acid ester of substrate 2 can significantly reduce the cytotoxic activity.

Three New Records of Mortierella Species Isolated from Crop Field Soil in Korea

Three new fungal species of the genus Mortierella, Mortierella zychae, Mortierella ambigua, and Mortierella indohii, have been reported in Korea. The fungi were encountered during a study on the fungal community of soil samples collected from different locations in Korea. The species were identified based on molecular and morphological analyses. This study presents detailed descriptions of the morphological observations and molecular phylogenetic analysis of these three fungi. All three species were found to be sensitive to triphenyltetrazolium chloride staining. M. zychae demonstrated the highest intensity of mycelial staining, indicating that this species has the highest potential to produce arachidonic acid of the three species. The staining results indicated that the newly recorded species could potentially be useful for arachidonic acid production.

Confirmation of Two Undescribed Fungal Species from Dokdo of Korea Based on Current Classification System Using Multi Loci

Using dilution plating method, 47 fungal isolates were obtained from a soil sample collected from Dokdo in the East Sea of Korea in 2013. In this study, two fungal isolates, EML-MFS30-1 and EML-DDSF4, were confirmed as undescribed species, Metarhizium guizhouense and Mortierella oligospora in Korea based on current classification system using multi loci including rDNA internal transcribed spacer, large subunit, small subunit, and beta-tubulin (BTUB) genes. Herein, detailed morphological descriptions on characters of the undescribed fungal species as well as their molecular phylogenetic status are provided with comparisons to related species.

Expression analysis for genes involved in arachidonic acid biosynthesis in Mortierella alpina CBS 754.68

The time courses for production of fungal biomass, lipid, phenolic and arachidonic acid (ARA) as well as expression of the genes involved in biosynthesis of ARA and lipid were examined in Mortierella alpina CBS 754.68. A significant increase in the arachidonic acid content in lipids that coincided with reduced levels of lipid was obtained. Reduced gene expression occurred presumably due to the steady reduction of carbon and nitrogen resources. However, these energy resources were inefficiently compensated by the breakdown of the accumulated lipids that in turn, induced up-regulated expression of the candidate genes. The results further indicated that the expression of the GLELO encoding gene is a rate-limiting step in the biosynthesis of ARA in the early growth phase.

First Report of Mortierella alpina (Mortierellaceae, Zygomycota) Isolated from Crop Field Soil in Korea

A new recorded species of Mortierella was recovered during the investigation of fungal communities in soil samples collected from different locations of Gangwon-do, Korea. The species was identified and described as Mortierella alpina on the basis of phylogenetic analysis of internal transcribed spacer sequences and morphological characteristics. This species has not been officially reported from Korea thus far.

Statistical optimization of arachidonic acid production by mortierella alpina CBS 754.68 in submerged fermentation

Arachidonic acid is an essential fatty acid in human nutrition. In the present study, production of arachidonic acid by Mortierella alpina CBS 754.68 was evaluated in submerged fermentation. The fermentation variables were selected in accordance with the Plackett-Burman [PB] design and further optimized via response surface methodology [RSM]. Five significant variables, namely glucose, yeast extract, temperature, agitation rate, and fermentation time were selected for the optimization studies. The statistical model was constructed via central composite design [CCD]. Following the optimization step arachidonic acid production increased by approximately 660.5%, when compared to the screening step. The results indicate that carrying out the fermentation under the conditions of glucose at 50 g/l; yeast extract at 14 g/l; temperature of 22°C; agitation rate of 180 rpm, and fermentation time of 8 days will increase the arachidonic acid production up to 3 g/l. Results show that the optimization of culture conditions could greatly increase arachidonic acid production by Mortierella alpina CBS 754.68

Progress in production of arachidonic acid by Mortierella alpina and genetic modification / 生物工程学报

Arachidonic acid, as an important polyunsaturated fatty acid, is identified as potential food additives or pharmaceuticals for their biological activities. In recent years, arachidonic acid production by Mortierella alpina is becoming a research highlight. The prophase relevant researches focused on the mutagenic breeding and fermentation optimization. With the depth of investigation, the advancement concerning pathway for the biosynthesis of arachidonic acid in Mortierella alpina has been made. In this review, we summarized the prophase work briefly. Mainly, we discussed the biosynthesis pathway of arachidonic acid, the key enzymes, the construction of transformation system and the genetic modification. In addition, the prospect of microorganism arachidonic acid production is put forward.

Biomass carbohydrates assimilation and lipid accumulation by Mortierella isabellina / 生物工程学报

Biomass carbohydrates assimilation and lipid accumulation by Mortierella isabellina M2 strain were investigated. Corn fiber hydrolysate was specially studied. The results showed M. isabellina M2 strain achieved growth and lipid accumulation while glucose, xylose, mannose and arabinose were introduced as single carbon source, respectively. When M. isabellina M2 strain was cultivated on corn fiber hydrolysate with 6% sugars concentration, the biomass reached 18.2 g/L, the lipid content of dry mycelia was 45.7%, and the lipid yield achieved 8.3 g/L. It provided a promising perspective for microbial oils production with biomass hydrolysates.

Mortierella isabellina lipid-soluble extracts improves lipid-induced renal injury in rats / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the effect of the Mortierella isabellina lipid-soluble extracts on improving hyperlipidemia and renal damage in lipid-induced renal injury rats.</p><p><b>METHODS</b>Thirty two male Wistar rats were divided into four groups: normal group (N), model group (M), the group treated with Mortierella isabellina lipid-soluble extracts (T) and Simvastatin (S). N group was fed a standard diet and the other groups were fed a high-fat diet. T and S groups were orally administered by Mortierella isabellina lipid-soluble extracts and Simvastatin respectively for 12 weeks. N and M groups were orally administered by the normal saline. Twenty four-hour urinary protein excretion was detected. The levels of serum ALB, TP, BUN, Scr, TG, TC, LDL and TG, TC, LDL in the kidney were measured at week 12. The histological change, ultrastructural change and lipid deposits in kidney were evaluated.</p><p><b>RESULTS</b>At the 12th week, compared with M group, twenty four hour urinary protein excretion and the levels of TC, LDL in serum and renal tissue in T and S group decreased (P < 0.01 or P < 0.05), but the levels of ALB and TP in T and S group increased markedly (P < 0.01). There was no significant difference of the levels of BUN, Scr, and TG among the four groups. The protein casts in renal tubules, inflammatory cell infiltration in tubulointerstitium as well as mild mesangial cell proliferation and expanded mesangial matrix were obseverd in M group. Electron micrograph demonstrated massive fusion of foot process of podocytes accompanied with rich lipid droplets in proximal tubular epithelial cells in M group.</p><p><b>CONCLUSION</b>The lipid-soluble extracts of Mortierella isabellina has beneficial effect on improving hypercholesterolemia and kidney damage in lipid-induced renal injury rat.</p>

Protoplast transformation of Mortierella isabellina with hygromycin B resistance plasmid PD4 / 生物工程学报

A strain Mortierella isabellina M6-22-4, which was sensitive to hygromycin B, was selected by treating parental spores with N-methyl-N' -Nitro-N-nitrosoguanidine (MNNG). Protoplasts of the strain Mortierella isabellina M6-22-4 were transformed successfully to hygromycin B resistance using the PD4 plasmid, which contains the Escherichia coli hph gene under the control of Mortierella alpina his H4.1 promoter. The PD4 plasmid was introduced by PEG/CaCl2 treatment. Transformation frequencies of 1.6 - 2.8 transformants/microg of DNA were achieved. Then they were successively incubated to non-selected PDA plates for 10 generations. About 31.6% transformants only from digested plasmid were mitotically stable and showed different hygromycin B resistance when they were incubated back to selection plates. The results of PCR and Southern analysis in three transformants indicated that the plasmid PD4 had been integrated into the fungal genome with 1 - 2 copies. This is the first report of Mortierella isabellina transformation system and supplies an important tool for further research into genetic manipulation of this filamentous fungus.

Hongos aislados desde suelos de bosques de Araucaria-Nothofagus después de un incendio en el Parque Nacional Tolhuaca / Fungi isolated from Araucaria-Nothofagu forests soils after a fire in National Park Tolhuaca

Se determinó después de dos años el efecto de un incendio natural sobre la población fúngica en un bosque Araucaria-Nothofagus, localizado en el Parque Nacional Tolhuaca. Las poblaciones fúngicas fueron aisladas desde el suelo de tres parcelas (una de control) y de dos profundidades por el método de las diluciones en agar extracto de malta y la identificación se hizo por el método de la taxonomía tradicional. Los hongos de las taxa Penicillium, Mortierella y Trichoderma fueron predominantes (79 por ciento en promedio) a la profundidad 0-20 cm, independiente de la época de muestreo y sin mayores diferencias taxonómicas en las 3 parcelas, después de los 2 años del incendio, siendo también dominantes (68.6 por ciento en promedio) en la profundidad 20-40 cm. Además, se observó un mayor número de taxa en la profundidad 0-20 cm (37 taxa) en comparación a los 20-40 cm (18 taxa).

After two years, the effect of a natural fire on the fungal population of an Araucaria-Nothofagus forest, located in Nacional Park Tolhuaca was assessed. Fungal populations were isolated from the soil of three plots and at two depths by the dilution in malt extract agarmethod. The identification was made by the traditional taxonomy method. Fungi of taxa Penicilium, Mortierella and Trichoderma are predominant (79 percent average) at 0- 20cm depth, regardless of the sampling time, and withouth mayor taxonomic differences in the three plots after the 2 years of fire and they are also dominant (68.6 percent average) at 20-40cm depth. Besides, a greater number of taxa at 0-20cm depth (37 taxa) was observed as compared to the 20-40cm (18 taxa) depth.

Expression of delta6-fatty acid desaturase gene from Mortierella alpina in Pichia pastoris / 生物工程学报

Gamma-linolenic acid (GLA, C18:3delta6 ,9,12), an essential polyunsaturated fatty acid, plays an important role in hormone regulation and fatty acid metabolization. Delta6-fatty acid desaturase (D6D) is the rate-limiting enzyme of the desaturation of linoleic acid (C18:2delta9,12) in the production of gamma-linolenic acid. A deficiency of GLA may have occurred when delta6-fatty acid desaturase activity decreases in aging, stress, diabetes, eczema, and some infections. To establish a new expression system for delta6-fatty acid desaturase gene in Pichia pastoris, which is an increasingly popular heterologous gene expression system, a gene encoding delta6-fatty acid desaturase from Mortieralla alpina was isolated by PCR amplification. The PCR product was then digested by EcoR I and Not I and subcloned into the intracellular expression vector pPIC3.5K to generate the recombinant vector pPIC3.5K-MA6. The resulting vector was linearized by Sac I and electroporated into P. pastoris SMD1168 (his- pep-) host cells. After electroporation, aliquots were spreaded on the MDS plates and incubated at 30 degrees C for three days until colonies appeared. Those transformants were subsequently screened for clones with high copy number by using the YPD plates containing G418. To identify the D6D constructs that were produced, chromosomal DNA of the transformants were prepared and used as template for PCR with the primer 5' AOX and 3' AOX. The PCR product of Mut+ recombinants was shown as a band of 1.38 kb of D6D gene and the product of 2.2 kb of AOX1 gene, while the product of Mut(s) transformants only was shown as a band of 1.38 kb of the D6D gene.To further confirm the transformants containing a functional D6D gene, the positive clones were selected and induced by methanol for expression. Those induced cultures were taken for analyses of the intracellular fatty acid composition by GC. The resultant chromatograms of fatty acid methyl esters showed that a novel peak was detected, which was not apparent in the case of control. Comparisons of the retention times of the newly yielded peaks with those of authentic standards have anticipated that the fatty acid is GLA. And this prospects was positively supported by definitive assignments of the compounds by GCMS analyses. Thus, the active delta6-fatty acid desaturase was expressed intracellularly in P. pastoris and gamma-linolenic acid reached 16.26% of the total fatty acid in recombinant P. pastoris strains. It was the first report about the expression of Mortieralla alpina D6D gene in P. pastoris.

Expression of Mortierella isabellina delta6-fatty acid desaturase gene in gamma-linolenic acid production in transgenic tobacco / 生物工程学报

Gamma-linolenic acid (GLA, C18:3delta6.9.12) is nutritional and important polyunsaturated fatty acid in human and animal diets. GLA play an important role in hormone regulation and fatty acid metabolization. Furthermore it is also the biological precursor of a group of molecules, including prostaglandins, leukotrienes and thromboxanes. Vast majority of oilseed crops do not produce GLA, but linoleic acid (LA, C18:2delta9.12) as its substrate. GLA is only produced by a small number of oilseed plants such as evening promrose ( Oenotheera spp.), borage (Borago officinalis) and etc. delta6-fatty acid desaturase (D6D) is the rate-limiting enzyme in the production of GLA. It can convert from linoleic acid to linolenic acid. To produce GLA in tobacco, plant expression vector was first constructed. To facilitate preparation of plant expression constructs, flanking Xba I and Bgl II restriction enzyme sites were added to the coding region of clone pTMICL6 by PCR amplification. pTMICL6 contains delta6-fatty acid desaturase gene cloned from Mortierella isabellina which is an oil-producing fugus. The PCR product was purified and subcloned into the plant expression vector pGA643 to generate the recombinant vector pGAMICL6 which contains the ORF of the D6D gene of Mortierella isabellina, together with regulatory elements consisting of the cauliflower mosaic virus 35S promoter and the nopaline synthase (nos) termination sequence. The plasmid pGAMICL6 was transformed into Agrobacterium tumefaciens strain LBA4404 by method of freeze thawing of liquid nitrogen. Transformants were selected by plating on YEB medium plates containing kanamycin and streptomycin and grown overnight at 28 degrees C, then transformants were further identified by PCR. The positive transformant containing the plant expression vector pGAMICL6 was transformed into tobacco ( Nicotiana tabacum cv. Xanthi) via Agrobacterium infection. Transgenic plants were selected on 100 microg/mL kanamycin. Plants were maintained in axionic culture under controlled conditions. Total nucleic acids were extracted and purified from anti-kanamycin transgenic tobacco and were analysed by PCR. 48 out of 80 transgenic plants were positive, in other words, transformation efficiency is 60% . This shows that Mortierella isabellina D6D gene is transformed into tobacco. Genomic DNA from PCR positive transgenic tobacco plants was digested with Hind III restriction enzyme and fractionated by agarose gel electrophoresis. Southern blotting was performed with strandard procedures for vacuum transfer of nucleic acids to nylon membrane. The probe was delta6-fatty acid desaturase gene from M. isabellina, which was labeled with DIG-dUTP via random-primed labeling. Hybridization and immumological detection were carried out the kit of DIG detection. The result shows single hybridizing bands in each of the transgenic tobacco plants DNA, but no hybridization was observed to non-transgenic tobacco. This indicates that delta6-fatty acid desaturase gene is integrated into the genome of transgenic tobacco. To provide further evidence that the introduction of the M. isabellina cDNA into the tobacco genome was responsible for the novel desaturation products, total RNA was isolated from GLA-positive transgenic tobacco plants via both PCR and Southern blotting and separated by electrophoresis through 1% formaldehyde agarose gel. Northern blotting including probe labeling, hybridization and detection was the same as Southern blotting in operation approach. A positive hybridization signal of identical mobility was obtained from RNA isolated from the transgenic tobacco plants, but not from the control tobacco plant. At last, total fatty acids extracted from the positive transgenic tobacco were analyzed by gas chromatography (GC) of methyl esters to confirm the transgenic tobacco containing a functional delta6-fatty acid desaturase gene. The result shows that two peaks were observed in the chromatogram of FAMes. GLA and octadecatetraenoic acid (OTA, C18:4delta6.9.12.15) respectively have 19.7% and 3.5% of the total fatty acids in the transgenic plant. The presence of both GLA and OTA indicates that the delta6-fatty acid desaturase used both linoleic acid and a-linolenic acid (ALA, C18:3delta6.9.12.15) as substrates, and this may be responsible for the decrease in ALA observed in the transgenic line. That was the first report about the expression of M. isabellina delta6-fatty acid desaturase gene in tobacco. All results mentioned above have laid the foundation of the thorough studying on an breeding transgenic oilseeds containing GLA to change the fatty acid composition of conventional oilseeds, it is significant to study on regulation mechanism of fatty acid desaturase.

A Case of Cutaneous Mucormycosis Developed in a Diabetic Patient with lnjection of Paraffin on the Face

Mucormycosis is an opportunistic fungal infection caused by the mucorales, which consist of the Rhizophus, Absidia, Mortierella, and Mucor. The diverse clinical forms of mucormycosis can be categorized as rhinocerebral, pulmonary, disseminated, gastrointestinal, cutaneous and miscellaneous. Although it can cause disease in healthy people, it most commonly affects patients who are diabetic or immunosup-pressed. Rarely, mucormycosis develops confined to the subcutaneous tissue. We experienced a case of cutaneous mucormycosis developed in a well-controlled diabetic patient who had previously injected foreign material on her face for an aesthetic purpose at the behest of an unlicensed physician. The patient had tissue necrosis and purulent discharge on her left cheek. Following histologic examination, we diagnosed the condition as cutaneous mucormycosis and treated it by wide excision combined with amphotericin B therapy. The disease was cured without any significant sequelae within 6 months of follow up. Cutaneous mucormycosis is rarely reported but it can cause facial deformity due to tissue necrosis, so that early diagnosis and appropriate treatment are important. Currently, foreign material injection for aesthetic purposes is still performed by unlicensed physicians. We present a case report concluding that one should consider the possibility of fungal infection in skin lesion in diabetic or other immunosup-pressed patients. We also emphasize the seriousness of illegal injection of foreign material.