Arachidonic Acid Yield Research Articles

Study on the regulation of Mortierella alpina morphology and high yield of arachidonic acid by metal ions

Study on the regulation of Mortierella alpina morphology and high yield of arachidonic acid by metal ions

Development of user-friendly protocols for single-cell arachidonic acid production in developing countries

Arachidonic acid (ARA) is a sorely needed nutrient and an effective and safe schistosomicide and tumoricide. Consumption levels are low in developing countries because of unawareness of its critical impact on health and limited commercial availability. The research work aims to encourage production in developing countries via providing user-friendly definitive protocols for polyunsaturated fatty acids production by the fungus Mortierella alpina and ARA isolation. Fatty acids content was determined by gas chromatography-flame ionization detector. The optimal cultivation conditions included culture medium to flask volume 1:5; shake-flask culture at 25 °C, then at 20 °C, and stationary aging at 10 °C, each over 7 days, while addition of trace minerals and vitamins was dispensable. The ARA yield attained >1.8 g per liter culture. Repeat experiments led to the selection of an optimal lipid extraction method from dried biomass, and efficacious urea complexation-based protocol for obtention of high ARA yield and purity.

Modular Metabolic Engineering of Mortierella alpina by the 2A Peptide Platform to Improve Arachidonic Acid Production.

Arachidonic acid (ARA) is an essential fatty acid in human nutrition. Mortierella alpina, a filamentous fungus, has been widely used for the production of ARA. Here, we report a modular engineering approach that systematically eliminates metabolic bottlenecks in the multigene elongase/desaturase pathway and has led to significant improvements of the ARA titer. The elongase/desaturase pathway in Mortierella alpina was recast into two modules, namely, push and pull modules, based on its function in the ARA synthesis. Combinatorial optimization of these two modules has balanced the production and consumption of intermediate metabolites. A 2A peptide-based facile assembly platform that can achieve multigene expression as a polycistron was first established. The platform was then applied to express the push and pull modules in Mortierella alpina. In the shake-flask fermentation, the lipid and ARA contents of the engineered strain MA5 were increased by 1.2-fold and 77.6%, respectively, resulting in about fivefold increase of the ARA yield. The final ARA titer reached 4.4 g L-1 in shake-flask fermentation. The modular engineering strategies presented in this study demonstrate a generalized approach for the engineering of cell factories in the production of valuable metabolites.

Redistributing Carbon Flux by Impairing Saccharide Synthesis to Enhance Lipid Yield in Oleaginous Fungus Mortierella alpina.

Increasing carbon flux toward target metabolites is important in improving microbial productivity and economic value. To improve the efficiency of lipid production in Mortierella alpina, we knocked down genes for trehalose-6-phosphate synthetase (Matps) and phosphoenolpyruvate carboxykinase (Mapepck) in the major pathways for saccharide synthesis. The knockdown of Matps reduced trehalose content by an average of 31.87%, while the knockdown of Mapepck reduced the total saccharide content by 28.6%, and both recombinant strains showed more than 20% increased lipid yield. Trehalose plays a vital role in stress resistance, but a higher polyunsaturated fatty acid-rich lipid content was found to partly compensate for the loss of trehalose after Matps knockdown. As compared with Matps knockdown, the knockdown of Mapepck gave better lipid production by bringing forward the time to maximum lipid yield by three days in a scale-up test. The arachidonic acid yield after the Mapepck knockdown reached 1.23 g/L, which was 39.9% higher than that of the original strain. The present research provided an efficient strategy for redistributing carbon flux among different metabolites and therefore promoted microbial lipid yield in a shorter fermentation period.

Statistical optimization of arachidonic acid synthesis by Mortierella alpina CBS 754.68 in a solid-state fermenter.

Arachidonic acid (ARA) is an omega‐6 fatty acid that plays a major role in human health. The present study optimizes the production of ARA by the soil fungus Mortierella alpina CBS 754.68 on oil cakes. In the first step, the best substrate was chosen from four oil cakes, namely soybean, sunflower, olive, and colza oil cakes, of which sunflower oil cake showed the highest yield. In the next step, screening tests were performed using the Plackett‐Burman design. Seven variables (substrate particle size, moisture content, time, temperature, yeast extract, glucose, and glutamate) were investigated (each taking values of +1 and −1). Among these variables, time, temperature, and substrate particle size significantly affected ARA production (p < .05), so they were further investigated in the optimization step. The optimal fermentation time, temperature, and substrate particle size calculated by response surface methodology were 8.75 days, 18.5°C, and 1.3 mm–1.7 mm, respectively. Under these conditions, M. alpina was predicted to produce 4.19 mg of ARA/g dry weight of substrate (DWS). The actual yield, determined in evaluation tests, was 4.48 ± 0.16 mg ARA/g DWS, which shows the accuracy of the model. In the final step, the effect of the aeration rate on producing ARA was investigated in a packed‐bed solid‐state fermenter under the determined optimal conditions. In this stage, the highest ARA yield was 10.13 ± 0.26 mg/g DWS, approximately double that of the optimization step, and this confirms that aeration increases ARA production by M. alpina.

SNF1β-Modulated Glucose Uptake and the Balance between Polyunsaturated Fatty Acids and Carbohydrates in Mortierella alpina.

A sucrose nonfermenting protein kinase 1 (SNF1) complex is an important metabolic regulator in fungi that is critical to cell metabolism and stress response. In this study, the role of an SNF1 β-subunit in the oleaginous fungus Mortierella alpina (MaSip2) was investigated. The MaSip2 contained a glycogen-binding domain and a conserved SNF1-complex interaction region; its transcriptional level during lipogenesis shared high consistency with a previously reported SNF1 γ-subunit (MaSnf4). Overexpression of MaSip2 in M. alpina significantly promoted glucose uptake and resulted in 34.1% increased total biomass, leading to 44.8% increased arachidonic acid yield after 7 day fermentation. MaSip2 also regulated the balance between polyunsaturated fatty acids and carbohydrates in M. alpina. Intracellular metabolite analysis revealed increased carbohydrate-related metabolite accumulation in MaSip2 overexpression strains. On the contrary, knockdown of MaSip2 increased the total fatty acid unsaturation degree, especially under low-temperature conditions. This research improved our knowledge of SNF1 complex in M. alpina and provided a target gene for enhancing glucose utilization and modulating fatty acid composition for better application of oleaginous fungi.

One step selection strategy for optimization of media to enhance arachidonic acid production under solid state fermentation

Mortierella alpina is the most researched fungus for production of arachidonic acid. Arachidonic acid inclusion in diet is gaining prominence due to its potential role in brain development and age-related disease conditions. The production mainly focuses on submerged fermentation with extensive use of gas chromatography which increases the cost of optimization. The present study focuses on the optimization of arachidonic acid production by M. alpina under solid-state fermentation and developing an effective mycelial staining method as an analytical tool to replace gas chromatography. The optimization method increased the Arachidonic acid yield (41.29 mg/gds) with the supplementation of 3.0% corn gluten, 3.0% flax seed oil and 0.625% of mineral mix-3 in a soybean meal based substrate at an optimum temperature of 25 °C and initial substrate moisture content of 70.0%. For deriving an effective analytical tool, correlation was made between the responses of statistically designed submerged fermentation and solid-state fermentation trials. The comparative regression analysis of the responses i.e., the mycelial Triphenyl tetrazolium chloride staining data and the arachidonic acid values revealed a 98.0% correlation. The submerged fermentation based mycelial staining was thus devised as an effective media optimization strategy for solid state fermentation based arachidonic acid production.

Biotransformation of Animal Fat-By Products into ARA-Enriched Fermented Bioproducts by Solid-State Fermentation of Mortierella alpina.

Solid-state fermentation (SSF) is a powerful fermentation technology for valorizing rest materials and by-products of different origin. Oleaginous Zygomycetes fungi are often used in SSF as an effective cell factory able to valorize a wide range of hydrophilic and hydrophobic substrates and produce lipid-enriched bioproducts. In this study, for the first time, the strain Mortierella alpina was used in SSF for the bioconversion of animal fat by-products into high value fermented bioproducts enriched with arachidonic acid (ARA). Two cereals-based matrixes mixed with four different concentrations of animal fat by-product were evaluated for finding optimal conditions of a fat-based SSF. All obtained fermented bioproducts were found to be enriched with ARA. The highest substrate utilization (25.8%) was reached for cornmeal and it was almost double than for the respective wheat bran samples. Similarly, total fatty acid content in a fermented bioproduct prepared on cornmeal is almost four times higher in contrast to wheat bran-based bioproduct. Although in general the addition of an animal fat by-product caused a gradual cessation of ARA yield in the obtained fermented bioproduct, the content of ARA in fungal biomass was higher. Thus, M. alpina CCF2861 effectively transformed exogenous fatty acids from animal fat substrate to ARA. Maximum yield of 32.1 mg of ARA/g of bioproduct was reached when using cornmeal mixed with 5% (w/w) of an animal fat by-product as substrate. Furthermore, implementation of attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy in characterization of obtained SSF bioproducts was successfully tested as an alternative tool for complex analysis, compared to traditional time-consuming methods.

Phytohormones as stimulators to improve arachidonic acid biosynthesis in Mortierella alpina

Phytohormones are chemical messengers that have a positive effect at low concentrations on the biosynthesis of high-value compounds. Therefore, the effects of phytohormones on lipid and arachidonic acid (ARA) biosynthesis in Mortierella alpina were investigated in this study. At proper concentrations, the stimulatory effects of phytohormones on lipid production were determined to be as follows: 6-benzyl adenine (BA) > indole-3-acetic acid (IAA) > furfuryl adenine (KT) > gibberellin (GA) > indole-3-butyric acid (IBA) > abscisic acid (ABA). The results show that in the presence of 15 mg L−1 BA, the best positive effect was obtained, in which the lipid and ARA yields of M. alpina increased by 20.34% and 29.17%, respectively. Surprisingly, there was no synergy between the addition of two cytokinins (KT and BA), while adding cytokinins (KT or BA) and auxin (IAA) inhibited the growth of M. alpina and the ARA yield decreased by approximately 64%. Additional studies, such as those involving enzyme activity detection and quantitative real time polymerase chain reaction were carried out to check the fatty acid and lipid biosynthesis when the phytohormones were present. The activity of the main NADPH-supplying enzyme, 6-phosphoglucose dehydrogenase (G6PDH), increased by 19.52%. Moreover, the transcription levels of fatty acid synthase (FAS), Δ9-desaturase, and diacylglycerolacyltransferase (DGAT) increased by 9.3, 9.6 and 7.7 times, respectively, when only one type of phytohormone was present, indicating the enhancement of fatty acid and lipid biosynthesis in M. alpina. This study demonstrates the potential application of phytohormones for improving ARA yields of M. alpina.

Heavy ion mutagenesis combined with triclosan screening provides a new strategy for improving the arachidonic acid yield in Mortierella alpina

BackgroundArachidonic acid (ARA), which is a ω-6 polyunsaturated fatty acid, has a wide range of biological activities and is an essential component of cellular membranes in some human tissues. Mortierella alpina is the best strain for industrial production of ARA. To increase its yield of arachidonic acid, heavy ion beam irradiation mutagenesis of Mortierella alpina was carried out in combination with triclosan and octyl gallate treatment.ResultsThe obtained mutant strain F-23 ultimately achieved an ARA yield of 5.26 g L− 1, which is 3.24 times higher than that of the wild-type strain. In addition, quantitative real-time PCR confirmed that the expression levels of fatty acid synthase (FAS), Δ5-desaturase, Δ6-desaturase, and Δ9-desaturase were all significantly up-regulated in the mutant F-23 strain, especially Δ6- and Δ9-desaturase, which were up-regulated 3- and 2-fold, respectively.ConclusionsThis study confirmed a feasible mutagenesis breeding strategy for improving ARA production and provided a mutant of Mortierella alpina with high ARA yield.

Manipulating the generation of reactive oxygen species through intermittent hypoxic stress for enhanced accumulation of arachidonic acid-rich lipids

The accumulation of reactive oxygen species (ROS) can induce oxidative damage which can be detrimental to microbial biomass and product yields. In this study, we used intermittent hypoxic stress (IHS) to eliminate the main source of ROS during the production of arachidonic acid (ARA)-rich lipids by Mortierella alpina. Biomass, lipid- and ARA concentration simultaneously reached their peak values, and the ARA yield increased by about 20% along with a lower level of ROS concentration. Under these conditions, more NADPH, substrate and energy may be available for lipid biosynthesis. The efficacy of this strategy was also confirmed in a bioreactor, with similar results. Interestingly, the total power required for stirring fell by about 4%, which may be of practical value for energy savings. This work offers new insights into the control of ROS generation in M. alpina and might be applicable to other microbial PUFAs producers.

Comparative assessment for efficiency of isolation of arachidonic acid (20:4n-6) from various sources

Methods of arachidonic acid (AA) isolation from animal and vegetal natural sources are considered. Traditional raw material as the lipids of beef liver and alternative source as the red alga Gracilaria vermiculophylla are tested. The AA was isolated by a sequence of known techniques for concentration of polyunsaturated fatty acids: sedimentation of saturated fatty acids in the form of their lithium salts, iodo-lactonization, and high performance liquid chromatography. All these techniques were applied for isolation of AA from the beef liver lipids, where the AA content did not exceed 7.7 % of total lipids, and the yield of AA with purity of 98.76 % was 50.8 %. The AA content in the algae lipids was significantly higher (&gt; 35 %), while the amount of n-3 polyunsaturated fatty acids was insignificant, so two techniques only were applied (sedimentation of lithium salts and liquid chromatography), and the yield of AA with purity of 97.60 % was 68.3 %. Thus, high-purity arachidonic acid could be isolated from the algae G. vermiculophylla using simple and reproducible procedure. Taking into account the high yield of AA, this method is economically feasible and is proposed for biochemical and medical researches.

Enhanced arachidonic acid production using a bioreactor culture of Mortierella alpina with a combined organic nitrogen source

The organic nitrogen source is one of the key factors affecting Mortierella alpina cell growth as well as arachidonic acid (ARA) production. The aim of the present work is to achieve an optimized recipe of organic nitrogen source for ARA production by M. alpina by testing four organic nitrogen sources. In the flasks, the results showed yeast extract or corn steep liquor were the most suitable sole nitrogen sources for biomass and ARA yield. In the bioreactor, a biomass of 17.5 g L−1 and an ARA yield of 2.7 g L−1 were achieved when using sole yeast extract, while cell autolysis was induced when using sole corn steep liquor; When using the combined nitrogen source with corn steep liquor and yeast extract at a 3:7 weight ratio, the biomass and ARA yield were significantly improved to 37 and 7.8 g L−1, respectively. This work evaluated whether using a mix of organic nitrogen sources could improve ARA production when scaling up from a flask to a bioreactor culture. The 3:7 ratio of corn steep liquor to yeast extract was quite favourable for large-scale ARA production, and as a result, this combination has great potential for improving fungal cultures.

Enhancing total fatty acids and arachidonic acid production by the red microalgae Porphyridium purpureum

This study investigated the effect of aeration rate and light intensity on biomass production and total fatty acids (TFA) accumulation by Porphyridium purpureum. The red microalgae is also known to accumulate considerable amount of arachidonic acid (ARA). In artificial seawater medium, the highest yield of TFA (473.44 mg/L) was obtained with the aeration rate of 3 L/min and light intensity of 165 µmol/m2s, whilst the highest yield of ARA (115.47 mg/L) was achieved with the aeration rate of 3 L/min and light intensity of 110 µmol/m2s. It was found that higher aeration rate led to more biomass and TFA/ARA production. However, higher light intensity could contribute to biomass accumulation, but it was adverse for TFA and ARA biosynthesis. By optimizing two operating factors (i.e., light intensity and aeration rate), TFA and ARA production by P. purpureum was significantly improved. This research provides a potential alternative means for producing ARA.

Improving arachidonic acid fermentation by Mortierella alpina through multistage temperature and aeration rate control in bioreactor

ABSTRACTEffective production of arachidonic acid (ARA) using Mortierella alpina was conducted in a 30-L airlift bioreactor. Varying the aeration rate and temperature significantly influenced cell morphology, cell growth, and ARA production, while the optimal aeration rate and temperature for cell growth and product formation were quite different. As a result, a two-stage aeration rate control strategy was constructed based on monitoring of cell morphology and ARA production under various aeration rate control levels (0.6–1.8 vvm). Using this strategy, ARA yield reached 4.7 g/L, an increase of 38.2% compared with the control (constant aeration rate control at 1.0 vvm). Dynamic temperature-control strategy was implemented based on the fermentation performance at various temperatures (13–28°C), with ARA level in total cellular lipid increased by 37.1% comparing to a constant-temperature control (25°C). On that basis, the combinatorial fermentation strategy of two-stage aeration rate control and dynamic temperature control was applied and ARA production achieved the highest level of 5.8 g/L.

Phosphate limitation promotes unsaturated fatty acids and arachidonic acid biosynthesis by microalgae Porphyridium purpureum.

Polyunsaturated fatty acids (PUFAs) are highly appreciated on their nutritive value for human health and aquaculture. P. purpureum, one of the red microalgae acknowledged as a promising accumulator of ARA, was chosen as the target algae in the present research. Effects of sodium bicarbonate (0.04-1.2g/L), temperature (25, 30 and 33°C) and phosphate (0.00-0.14g/L) on biomass yield, total fatty acids (TFA) and arachidonic acid (ARA) accumulation were investigated systemically. NaHCO3 dose of 0.8g/L and moderate temperature of 30°C were preferred. In addition, TFA and ARA production were significantly enhanced by an appropriate concentration of phosphate, and the highest TFA yield of 666.38mg/L and ARA yield of 159.74mg/L were obtained at a phosphate concentration of 0.035g/L. Interestingly, with phosphate concentration continuing to fall, UFA/TFA and ARA/EPA ratios were increased accordingly, suggesting that phosphate limitation promoted unsaturated fatty acids and arachidonic acid biosynthesis. Low concentration of phosphate may be favored to increase the enzymatic activities of ∆6-desaturase, which played a key role in catalyzing the conversion of C16:0 to C18:2, and thus the selectivity of UFA increased. Meanwhile, the increase of ARA selectivity could be attributed to ω6 pathway promotion and ∆17-desaturase activity inhibition with phosphate limitation. Phosphate limitation strategy enhanced unsaturated fatty acids and ARA biosynthesis in P. purpureum, and can be applied in commercial scale manufacturing and commercialization of ARA.

The effect of pH, aeration, and temperature on arachidonic acid synthesis by Mortierella alpina

The effects of pH, aeration, and temperature on the growth of fungal strain Mortierella alpina LPM-301 and the synthesis of lipids and arachidonic acid in glycerol-containing medium were studied. Arachidonic acid production in the stationary growth phase was found to depend considerably on the pH value; it reached the optimum at pH 6.0 and was irreversibly inhibited at a pH of 3.0. The PO2 values in a range from 10 to 50% showed no marked effect on mycelium growth or the synthesis of lipids and arachidonic acid. The temperature optimum for arachidonic acid production was 20-22 degrees C. Under continuous cultivation, the amount of arachidonic acid reached 29.8% of lipids and 7.4% of biomass. The arachidonic acid yield from the glycerol consumed was 4.1% by mass and 8.8% by energy. It is suggested that arachidonic acid synthesis at an unfavorable pH and elevated temperatures was limited by the activity of A-12-desaturase and by the conversion of linoleic to arachidonic acid, respectively.

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 5.09g/L, a 40.61% increase over the original strain (3.62g/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 (6.82g/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.

Pythium irregulare Fermentation to Produce Arachidonic Acid (ARA) and Eicosapentaenoic Acid (EPA) Using Soybean Processing Co-products as Substrates

Arachidonic acid (ARA) and eicosapentaenoic acid (EPA) were produced by Pythium irregulare fungus using soybean cotyledon fiber and soy skim, two co-products from soybean aqueous processing, as substrates in different fermentation systems. Parameters such as moisture content, substrate glucose addition, incubation time, and vegetable oil supplementation were found to be important in solid-state fermentation (SSF) of soybean fiber, which is to be used as animal feed with enriched long-chain polyunsaturated fatty acids (PUFA). Soybean fiber with 8 % (dwb) glucose supplementation for a 7-day SSF produced 1.3 mg of ARA and 1.6 mg of EPA in 1 g of dried substrate. When soy skim was used as substrate for submerged fermentation, total ARA yield of 125.7 mg/L and EPA yield of 92.4 mg/L were achieved with the supplementation of 7 % (w/v) soybean oil. This study demonstrates that the values of soybean fiber and soy skim co-products could be enhanced through the long-chain PUFA production by fermentation.

Development of a Defined Medium for Arachidonic Acid Production by Mortierella alpina Using a Visualization Method

Defined medium for arachidonic acid (ARA) production by Mortierella alpina was optimized for its metabolomics study. For this purpose, a visualization method (VM) was applied for the first time. Experiments were designed according to the uniform design with four factors (concentrations of glucose, NaNO(3), KH(2)PO(4) and MgSO(4)·7H(2)O) for each at nine levels. Dry cell weight (DCW), ARA yield in DCW [percent (w/w)] and ARA content in total fatty acids [percent (w/w)] were considered as the three objectives. Optimization of single-objective function and multi-objective function of two objectives and three objectives was attempted. Optimal DCW, ARA yield and ARA content were predicted to occur in a medium that contained (grams per litre): glucose 35, NaNO(3) 1, KH(2)PO(4) 7.5 and MgSO(4)·7H(2)O 2.6. Upon verification, the average tested DCW (12.95 g/l), ARA yield (18.89 %) and ARA content (42.36 %) were fairly close to the predicted values (12.88 g/l, 9.68 % and 35.57 %, respectively). Moreover, DCW, ARA yield and ARA content from the optimum medium increased by 35.68, 47.23 and 30.90 % compared with control, respectively, indicating that VM had succeeded in exploiting the biomass growth and ARA production by M. alpina.