Production of arachidonic acid by Mortierella alpina using wastes from potato chips industry.

AIMS: This research aimed to determine the potential use of wastes from the potato chips industry as a carbon source to develop an economical culture medium for the production of biomass, lipids and arachidonic acid (ARA) by Mortierella alpina. METHODS AND RESULTS: A synthetic culture medium was optimized using a Plackett-Burman and central composite rotatable design, and used as a base to evaluate and characterize the potential use of wastes from the potato chips industry as carbon sources for the production of biomass, lipids and ARA by M. alpina. The waste was selected among other solid and liquid hydrolysed residues/by-products, and local low-cost alternatives for nitrogen sources were also evaluated. After 6 days of fermentation, the biomass concentration reached 20 g l-1 with 40% of total lipids, and a 35% ARA content in the lipids fraction. Savings in production were calculated using a sensitivity analysis for the alternative culture medium in different scenarios. CONCLUSIONS: This study showed a 7% savings in culture media expenses in the production of ARA-enriched biomass of M. alpina, compared to the conventional synthetic culture medium, when waste from the potato chips industry was used as an alternative source of carbon and macro/microelements, supplemented with a low-cost yeast extract alternative. SIGNIFICANCE AND IMPACT OF THE STUDY: The demonstration of the use of potato chips wastes as a low-cost carbon source for the biomass, lipids and ARA production, suggesting an eco-friendly alternative for the use of agri-food wastes for valuable metabolites production.

Ultra Performance Liquid Chromatography-Q Exactive Orbitrap/Mass Spectrometry-Based Lipidomics Reveals the Influence of Nitrogen Sources on Lipid Biosynthesis of Mortierella alpina.

The objective of the present study was to reveal the effects of four types of nitrogen sources (soymeal, yeast extract, KNO3, and ammonium tartrate) on the lipid metabolism of the oleaginous fungus Mortierella alpina using untargeted lipidomics, targeted fatty acid, and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis. Our results showed clear differences in the contents and compositions of lipids between four types of nitrogen sources. Soymeal and ammonium tartrate supplementation favored the accumulation of triglycerides with arachidonic acid (ARA) and C16-18 fatty acids, respectively. These results were further validated by our targeted fatty acid analysis. RT-qPCR analysis of related genes in M. alpina between the four nitrogen source conditions found that soymeal supplementation dramatically increased the expression of GPAT, ELOVL, and Δ12/Δ6 desaturase. Our findings provided new insights into the regulation of lipid biosynthesis in M. alpina and potential avenues for genetic manipulation and highlighted the importance of an optimal nitrogen source for ARA-rich oil production.

How nitrogen sources influence Mortierella alpina aging: From the lipid droplet proteome to the whole-cell proteome and metabolome.

Arachidonic acid (ARA) is a valuable polyunsaturated fatty acid produced by Mortierella alpina. Although some strategies such as nitrogen supplementation have shown the potential to affect the aging of M. alpina in ways which enable it to produce more ARA, the underlying mechanism remains elusive. Herein, we conducted a systematical analysis of the lipid droplet proteome, as well as the whole-cell proteome and metabolome, in order to elucidate how and why two different nitrogen sources (KNO3 and urea) affect the aging of M. alpina and the corresponding ARA concentration. We found that KNO3 promoted the ARA concentration, while urea accelerated lipid consumption and stimulated the decomposition of mycelia. Although both KNO3 and urea activated carbohydrate metabolic pathways, KNO3 exerted a stronger promoting effect on the pentose phosphate pathway and induced the lipid droplets to participate in the citrate-pyruvate cycle. The activities of malic enzyme and isocitrate dehydrogenase were also promoted more by KNO3. These pathways provided additional substrates and reducing power for ARA synthesis and ROS elimination. Accordingly, since urea showed a weaker promotion of the related pathways, it caused a depression of the antioxidant system and a consequent increase of ROS. These findings facilitate the design of nitrogen supplementation strategies to achieve higher ARA concentrations, and provide guidance for deciphering the mechanisms of similar aging phenomena in other oleaginous microorganisms. SIGNIFICANCE: Polyunsaturated fatty acids such as arachidonic acid (ARA) are valuable nutrients, which play important roles in preventing numerous diseases and facilitating development. Although it has been found for years that ARA production will be increased in the aging process of Mortierella alpina (M. alpina) and nitrogen sources are involved in this process, the underlying mechanism for this phenomenon remains unknown. In this work, we used the subcellular proteomics, whole-cell proteomics and metabolomics methods to explore the mechanisms by which two different nitrogen (KNO3 and urea) affected the aging process of M. alpina. Finally, we gave some new insights for the mechanisms mentioned above. This finding will fuel the technology developments for the ARA production using microbes.

Application of a delta-6 desaturase with α-linolenic acid preference on eicosapentaenoic acid production in Mortierella alpina.

BACKGROUND: Delta-6 desaturase (FADS6) is a key bifunctional enzyme desaturating linoleic acid (LA) or α-linolenic acid (ALA) in the biosynthesis of polyunsaturated fatty acids (PUFAs). In previous work, we analyzed the substrate specificity of two FADS6 enzymes from Mortierella alpina ATCC 32222 (MaFADS6) and Micromonas pusilla CCMP1545 (MpFADS6), which showed preference for LA and ALA, respectively. We also clarified the PUFA profiles in M. alpina, where these lipids were synthesized mainly via the ω6 pathway and rarely via the ω3 pathway and as a result contained low ALA and eicosapentaenoic acid (EPA) levels. RESULT: To enhance EPA production in M. alpina by favoring the ω3 pathway, a plasmid harboring the MpFADS6 gene was constructed and overexpressed in a uracil-auxotrophic strain of M. alpina using the Agrobacterium tumefaciens-mediated transformation (ATMT) method. Our results revealed that the EPA production reached 80.0 ± 15.0 and 90.4 ± 9.7 mg/L in MpFADS6 transformants grown at 28 and at 12 °C, respectively. To raise the level of ALA, free form fatty acid was used as exogenous substrate, which increased the EPA production up to 114.5 ± 12.4 mg/L. To reduce the cost of EPA production in M. alpina, peony seed oil (PSO) and peony seed meal (PSM) were used as source of ALA, and EPA production was improved to 149.3 ± 7.8 and 515.29 ± 32.66 mg/L by supplementing with 0.1 % PSO and 50 g/L PSM, respectively. The EPA yield was further increased to 588.5 ± 29.6 mg/L in a 5-L bioreactor, which resulted in a 26.2-fold increase compared to EPA production in wild-type M. alpina. In this work, we have significantly enhanced EPA production through overexpression of a FADS6 desaturase with preference for ALA, combined with supplementation of its substrate. CONCLUSION: An ALA-preferring FADS6 from M. pusilla CCMP1545 was applied to enhance EPA production in M. alpina. By exogenous addition of peony seed oil or peony seed meal, EPA production was further increased in flasks and fermenters. This research also highlights the value of peony seed meal which can be converted to a high value-added product containing EPA, and as a way to increase the EPA/AA ratio in M. alpina.

Regio- and stereo-selective hydroxylations of ingenane diterpenoids by Mortierella ramanniana and Gibberella fujikuroi.

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 IC50 values being 35.59 ± 5.37 µmol·L-1 (Caco-2), 24.04 ± 4.70 µmol·L-1 (MCF-7), and 22.24 ± 5.19 µmol·L-1 (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.

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.

Evaluation of the effect of process variables on the fatty acid profile of single cell oil produced by Mortierella using solid-state fermentation.

This article reviews some of the aspects of single cell oil (SCO) production using solid-state fermentation (SSF) by fungi of the genus Mortierella. This article provides an overview of the advantages of SSF for SCO formation by the aforementioned fungus and demonstrates that the content of the polyunsaturated fatty acids (PUFA) depend on the type of fermentation media and culture conditions. Process variables that influence lipid accumulation by Mortierella spp. and the profile of the fatty acids are discussed, including incubation temperature, time, aeration, growth phase of the mycelium, particle size of the substrate, carbon to nitrogen ratio, initial moisture content and pH as well as supplementation of the substrate with nitrogen and oil. Finally, the article highlights future research trends for the scaled-up production of PUFAs in SSF.

Arachidonic acid production by Mortierella alpina using raw crop materials.

BACKGROUND: Arachidonic acid (ARA) is one of the three essential fatty acids, and it is important for human body to keep healthy and is widely used. At present, expensive materials such as glucose and yeast extract are generally reported to be optimal for ARA production. A new cost-effective fermentation process including cheaper material for ARA production is of great significance. METHODS: Feasibility of using corn meal and powdered soybean for fungal growth and lipid accumulation was evaluated by means of single factor test. N-hexadecane concentration was optimized, and the effect of temperature on biomass and ARA content was examined. RESULTS: Mortierella alpina made better use of the aforementioned material as carbon and nitrogen sources for both hyphae growth and ARA production compared with glucose and yeast extract. Maximal levels of 10.9 g/L ARA and 26.1 g/L total lipids were obtained when 66 g/L corn meal, 54 g/L soybean meal and 6% (v/v) n-hexadecane were supplemented. A temperature-shift strategy involved three steps, namely, 30°C (3 days) - 25°C (4 days) - 20°C (4 days), which further improved ARA production by 24.7%. CONCLUSIONS: Several factors such as carbon and nitrogen sources, temperature and dissolved oxygen had great influence on biomass and microbial oil production. Mortierella alpina preferred corn and soybean meal compared with glucose and yeast extract, which would surely alleviate the high cost of ARA production. Based on this study, the new process is both low cost and practicable.

Production of 8-prenylnaringenin from isoxanthohumol through biotransformation by fungi cells.

8-Prenylnaringenin (8PN), which presents in hop, enjoys fame as the most potential phytoestrogen. Although a number of health effects are attributed to 8PN, few reports are available about the production of it. In this work, screening of fungi to efficiently transform isoxanthohumol (IXN) into 8PN was designed. The biotransformation of IXN was significantly observed in Eupenicillium javanicum, Cunninghamella blakesleana, and Ceriporiopsis subvermispora under five kinds of transformation conditions. As a comparative result of IXN transformation, E. javanicum was the optimal biocatalyst to produce 8PN. Transformation caused by growing precultured fungal mycelia, a process designated as G2, was a favorable condition for IXN transformation in view of the yield of 8PN. The possible transformation pathway of 8PN bioproduction is postulated in this work. The construction of fungus and transformation mode derived from the current work is viable and an alternative procedure for 8PN formation.

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.