Enhanced arachidonic acid production from Mortierella alpina combining atmospheric and room temperature plasma (ARTP) and diethyl sulfate treatments.

To obtain mutant strains with higher arachidonic acid (ARA) yields, the oleaginous fungus Mortierella alpina was mutated using atmospheric and room temperature plasma (ARTP) coupled with diethyl sulfate (DES). A visual compound filter operation was used in which a screening medium was supplemented with cerulenin, an inhibitor of fatty acid synthase (FAS), and triphenyltetrazolium chloride (TTC). The mutant strain D20 with an ARA production of 5.09 g/L, a 40.61% increase over the original strain (3.62 g/L), was isolated. The relative ARA content increased from 38.99% to 45.64% of total fatty acids. After optimizing fermentation conditions, the maximum ARA yield (6.82 g/L) for strain D20 was obtained in shake flasks. This work provides an appropriate strategy for obtaining high ARA-yield strains by conventional random mutation methods with an efficient screening assay.

Prolonging storage time of baby ginger by using a sand-based storage medium and essential oil treatment.

Wilt and rot occur readily during storage of baby ginger because of its tender skin and high moisture content (MC). A storage medium, which consisted of sand, 20% water, and 3.75% super absorbent polymers delayed weight loss and loss of firmness at 12 °C and 90% relative humidity. Microorganisms were isolated and purified from decayed rhizomes; among these, 3 fungi were identified as pathogens. The results of 18S rDNA sequence analysis showed that these fungi belonged to Penicillium, Fusarium, and Mortierella genera. The use of essential oil for controlling these pathogens was then investigated in vitro. Essential oils extracted from Cinnamomum zeylanicum (cinnamon) and Thymus vulgaris (thyme) completely inhibited the growth of all of the above pathogens at a concentration of 2000 ppm. Cinnamon oil showed higher antifungal activity in the drug sensitivity test with minimal fungicidal concentration (<500 ppm against all pathogens). In the in vivo test, cinnamon fumigation at a concentration of 500 ppm reduced infection rates of Penicillium, Fusarium, and Mortierella by 50.3%, 54.3%, and 60.7%, respectively. We recommended cinnamon oil fumigation combined with medium storage at 12 °C as an integrated approach to baby ginger storage.

Metabolism and synthesis of lipids in the polyunsaturated fatty acid-producing fungus Mortierella alliacea.

The fungal strain Mortierella alliacea YN-15 is a promising industrial producer of polyunsaturated fatty acids (PUFAs), in particular arachidonic acid. In order to more efficiently produce PUFAs, the metabolism of an externally supplied plant oil, α-linolenic acid (ALA)-rich linseed triacylglycerol (TAG), was examined, and time-dependent changes in the composition of its lipid and fatty acid metabolites were traced. Addition of linseed TAG to growing cultures resulted in a transient increase in extracellular 1,2-diacylglycerol (DAG), and even more so of 1,3-DAG, in the mycelia. This was followed by a decrease in both DAGs and an increase in TAG. Eicosapentaenoic acid (EPA), a desaturated and elongated product of ALA, accumulated to a greater extent in cellular phospholipids than in neutral lipids. Moreover, the addition of ALA in free fatty acid form to the culture led to the generation of EPA. However, EPA production was not observed upon addition of ALA-rich 1,2- or 1,3-DAG, indicating that fatty acids released from exogenous lipids were used for resynthesis of mycelial TAG. These results suggested that TAG might be hydrolyzed by extracellular lipases, whereas its synthesis might be catalyzed by intracellular enzymes. Appropriate regulation of such enzymes might be an effective strategy to enhance PUFA production under plant oil supplementation.

Repression of reserve lipid turnover in Cunninghamella echinulata and Mortierella isabellina cultivated in multiple-limited media.

AIMS: To study patterns of reserve lipid biosynthesis and turnover (degradation) in two oleaginous Zygomycetes, namely Cunninghamella echinulata and Mortierella isabellina under various growth conditions. Fatty acid composition of the reserve lipid of both strains was also studied in all growth steps. METHODS AND RESULTS: Cunninghamella echinulata and Mortierella isabellina were grown in carbon-excess batch cultures. In the investigated strains, accumulation of reserve lipid occurred only when the activity of both NAD(+)-isocitrate dehydrogenase (ICDH) and NADP(+)-ICDH were not detectable in the cell-free extract. Specifically, in C. echinulata, NAD(+)-ICDH activity was detected even after depletion of ammonium nitrogen in the medium, resulting in a delay of the initiation of lipid accumulation period. On the contrary, in M. isabellina, lipid accumulation occurred simultaneously with ammonium nitrogen exhaustion in the growth medium, as the activity of both NAD(+)- and NADP(+)-ICDH were not detectable after nitrogen depletion. In C. echinulata reserve lipid was not degraded after glucose had been exhausted. Supplementations of the medium with Fe(3+), yeast extract or Mg(2+) induced, however, reserve lipid breakdown and formation of lipid-free material. In M. isabellina after glucose exhaustion, notable lipid degradation occurred, accompanied by a significant lipid-free material biosynthesis. Nevertheless, in multiple-limited media, in which Mg(2+) or yeast extract, besides carbon and nitrogen, were limiting nutrients, reserve lipid breakdown was repressed. In both strains, the quantity of gamma-linolenic acid (GLA) in the reserve lipids [varying between 9 and 16% (w/w) in C. echinulata and 1.5-4.5% (w/w) in M. isabellina] was proportional to lipid-free biomass. CONCLUSIONS: Lipid accumulation period in Zygomycetes is initiated by the attenuation of ICDH activity in the mycelium while the regulation of ICDH from ammonium nitrogen is strain specific. While a single nitrogen limitation was enough to induce lipid accumulation, however, multiple limitations were needed in order to repress lipid turnover in oleaginous Zygomycetes. As for GLA, its biosynthesis in the mycelium seemed proportional to lipid-free biomass synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Several nutrients are indispensable for functioning the mechanisms involved in the mobilization of reserve lipid in oleaginous moulds. Therefore, reserve lipid turnover in oleaginous moulds could be repressed in multiple-limited media.

Improved arachidonic acids production from the fungus Mortierella alpina by glutamate supplementation.

The effect of various concentrations of glutamate on arachidonic acid (AA) production from Mortierella alpina in shaker flask culture was studied. Glutamate supplementation promoted Mortierella growth, accelerated substrate metabolism, and increased AA production, and a concentration of 0.8 g/l glutamate resulted in the greatest AA yield (1.41 g/l). In 10 l airlift stirred fermenter culture, AA yield in the cultures exposed to 0.8 g/l glutamate was also greater than that in the control (0.56 g/l).

New antifungal and cytotoxic steroidal saponins from the bulbs of an elephant garlic mutant.

Saponins in bulbs of a mutant of elephant garlic were investigated, and three new steroidal saponins named yayoisaponins A-C were obtained together with the known dioscin and aginoside. Their structures, including the relative stereochemistry, were elucidated by spectral data interpretation, while the absolute stereochemistry of the sugar moieties was assigned on the basis of a chiral gas chromatographic analysis of the acid hydrolysates. Yayoisaponins A-C and aginoside exhibited not only in vitro cytotoxicity against P388 cells at 2.1 micrograms/ml, but also antifungal activity against Mortierella ramanniana at 10 micrograms/disk.