Γ-linolenic Acid Production Research Articles

Simultaneous production of γ-linolenic acid and carotenoids by a novel microalgal strain isolated from the underexplored habitat of intermittent streams

This study provides an in-depth investigation of biotechnological potential of a novel strain of microalga Pseudomuriella engadinensis KASU1, isolated from intermittent stream in the temperate region of Central Europe. Survival in drying environment of the intermittent streams requires strategies that are often related to the production of unique compounds. The newly isolated strain grows fast (doubling time as fast as 12 h) and robustly under a range of nitrogen concentrations (1.76–17.6 mM), light intensities (100–1200 μE m−2 s−1) and temperatures (20–25 °C), as well as under CO2 levels as high as present in the flue gas (up to 10% CO2). P. engadinensis produces essential fatty acids such as oleic acid, linoleic acid and γ-linolenic acid, and carotenoids such as β-carotene and lutein. γ-Linolenic acid was produced with the highest rate reported for microalgae (44 mg L−1 d−1), and lutein was produced with significantly higher rate compared to marigold petals traditionally used for its production. P. engadinensis showed high potential for nutraceutical production and waste gases treatment since it fixed up to 83 mg carbon L−1 d−1.

Evaluation of Different Standard Amino Acids to Enhance the Biomass, Lipid, Fatty Acid, and γ-Linolenic Acid Production in Rhizomucor pusillus and Mucor circinelloides.

In this study, 18 standard amino acids were tested as a single nitrogen source on biomass, total lipid, total fatty acid (TFA) production, and yield of γ-linolenic acid (GLA) in Rhizomucor pusillus AUMC 11616.A and Mucor circinelloides AUMC 6696.A isolated from unusual habitats. Grown for 4 days at 28°C, shaking at 150 rpm, the maximum fungal biomass for AUMC 6696.A was 14.6 ± 0.2 g/L with arginine and 13.68 ± 0.1 g/L with asparagine, when these amino acids were used as single nitrogen sources, while AUMC 11616.A maximum biomass was 10.73 ± 0.8 g/L with glycine and 9.44 ± 0.6 g/L with valine. These were significantly higher than the ammonium nitrate control (p < 0.05). The highest levels of TFA were achieved with glycine for AUMC 11616.A, 26.2 ± 0.8% w/w of cell dry weight, and glutamic acid for AUMC 6696.A, 23.1 ± 1.3%. The highest GLA yield was seen with proline for AUMC 11616.A, 13.4 ± 0.6% w/w of TFA, and tryptophan for AUMC 6696.A, 12.8 ± 0.3%, which were 38% and 25% higher than the ammonium tartrate control. The effects of environmental factors such as temperature, pH, fermentation time, and agitation speed on biomass, total lipids, TFA, and GLA concentration of the target strains have also been investigated. Our results demonstrated that nitrogen assimilation through amino acid metabolism, as well as the use of glucose as a carbon source and abiotic factors, are integral to increasing the oleaginicity of tested strains. Few studies have addressed the role of amino acids in fermentation media, and this study sheds light on R. pusillus and M. circinelloides as promising candidates for the potential applications of amino acids as nitrogen sources in the production of lipids.

Improved γ-Linolenic Acid Production from Cellulose in Mucor circinelloides via Coexpression of Cellobiohydrolase and Delta-6 Desaturase.

The present study was aimed at facilitating the production of γ-linolenic acid (GLA) from the cellulosic substrate with the engineered oleaginous fungus Mucor circinelloides WJ11. Here, the homologous recombination technology was used to overexpress the cellobiohydrolase (CBH2) derived from Trichoderma longibrachiatum and the original delta-6 fatty acid desaturase (D6) in M. circinelloides to construct genetically engineered strains capable of effectively using cellulose to enhance GLA synthesis. When cultivated in modified K&R medium supplemented with microcrystalline cellulose, the CBH2 and D6 coexpressing strains led to increases in the biomass (up to 12.8 g/L) and lipid yield (up to 3.7 g/L) of 87% and 2.4-fold, respectively, compared to that of the control strain. Notably, when CBH2 and D6 were coexpressed in M. circinelloides, the yield of GLA reached 608 mg/L, which was a dramatic increase of 3.9-fold compared to that of the control strain. This is the first report on promoting the GLA production from the cellulosic substrate via coexpression of CBH2 and delta-6 desaturase. This work provides a theoretical basis for efficient transformation from the cellulosic substrate to functional GLA by CBH2 and D6 coexpressing strains, which might play a positive role in promoting the sustainable development of biological industry.

Reengineering lipid biosynthetic pathways of Aspergillus oryzae for enhanced production of γ-linolenic acid and dihomo-γ-linolenic acid

Biological significance of 18-carbon polyunsaturated fatty acids, γ-linolenic acid (GLA; C18:3 n-6) and dihomo-γ-linolenic acid (DGLA; C20:3 n-6) has gained much attention in the systematic development of optimized strains for industrial applications. In this work, a n-6 PUFAs-producing strain of Aspergillus oryzae was generated by manipulating metabolic reactions in fatty acid modification and triacylglycerol biosynthesis. The codon-optimized genes coding for Δ6-desaturase and Δ6-elongase of Pythium sp., and diacylglycerol acyltransferase 2 (mMaDGAT2) of Mortierella alpina were co-transformed in a single vector into A. oryzae BCC14614, yielding strain TD6E6-DGAT2. Comparative phenotypic analysis showed that a 70% increase of lipid titer was found in the engineered strain, which was a result of a significant increase in triacylglycerol (TAG) content (52.0 ± 1.8% of total lipids), and corresponded to the increased size of lipid particles observed in the fungal cells. Interestingly, the proportions of GLA and DGLA in neutral lipids of the engineered strain were similar, with the highest titers obtained in the high C:N culture (29:0; 6% glucose) during the lipid-accumulating stage of growth. Time-course expression analysis of the engineered strain revealed transcriptional control of TAG biosynthesis through a co-operation between the native DGAT2 of A. oryzae and the transformed mMaDGAT2.

Optimization of Process Parameters for the Production of γ-Linolenic Acid by Cunninghamella elegans CFR C07 in Submerged Fermentation.

The production of γ-linolenic acid (GLA) by the fungus Cunninghamella elegans CFR C07 in submerged fermentation was studied. Culture parameters such as carbon source and incubation time were optimized. Four different extraction methods using solvents with acid washed sand, glass beads, lyophilization and Soxhlet extraction were evaluated for improved extraction of lipids from the fungal biomass after fermentation. The GLA production was initially optimized in 250-mL flask and the process was demonstrated in a 3-litre fermentor. The maximum GLA production was 882 mg/L in shake flask culture and 733 mg/L in the fermentor. The study shows that Cunninghamella elegans CFR C07 is a potent organism for the production of GLA under submerged conditions.

Engineering Yarrowia lipolytica for efficient γ-linolenic acid production

γ-Linolenic acid (GLA) has various well-documented beneficial physiological effects and high biological significance. Because the natural supply of GLA is insufficient, microbial GLA production is a promising method for pharmaceutical and nutraceutical purposes. To establish and develop a biotechnological process for GLA production by Yarrowia lipolytica, the codon-optimized △6-desaturase from Mortierella alpina was introduced into this yeast under the control of the strong hp4d promoter. A recombinant Y. lipolytica strain was constructed, which produced 4.6% GLA in total fatty acids. By using a temperature-shift strategy of cultivation, consisting in preliminary growth at 28°C followed by 6-day culture at 20°C, optimal levels of dry cell weight (DCW), lipid content and GLA concentration were obtained from the recombinant strain: 18.55g/L, 1.16g/L and 71.6mg/L, respectively. These DCW, lipid and GLA values were respectively 25.7%, 19.6% and 60.9% higher than those obtained in the control cultivation experiment at the standard constant temperature of 28°C. This work demonstrates the excellent capacity of Y. lipolytica for GLA production, by combining metabolic engineering with a temperature-shift strategy.

Production of γ-linolenic acid and stearidonic acid by Synechococcus sp. PCC7002 containing cyanobacterial fatty acid desaturase genes

Genetic modification is useful for improving the nutritional qualities of cyanobacteria. To increase the total unsaturated fatty acid content, along with the ratio of ω-3/ω-6 fatty acids, genetic engineering can be used to modify fatty acid metabolism. Synechococcus sp. PCC7002, a fast-growing cyanobacterium, does not contain a Δ6 desaturase gene and is therefore unable to synthesize γ-linolenic acid (GLA) and stearidonic acid (SDA), which are important in human health. In this work, we constructed recombinant vectors Syd6D, Syd15D and Syd6Dd15D to express the Δ15 desaturase and Δ6 desaturase genes from Synechocystis PCC6803 in Synechococcus sp. PCC7002, with the aim of expressing polyunsaturated fatty acids. Overexpression of the Δ15 desaturase gene in Synechococcus resulted in 5.4 times greater accumulation of α-linolenic acid compared with the wild-type while Δ6 desaturase gene expression produced both GLA and SDA. Co-expression of the two genes resulted in low-level accumulation of GLA but much larger amounts of SDA, accounting for as much to 11.64% of the total fatty acid content.

Optimization of medium components using orthogonal arrays for γ-Linolenic acid production by Spirulina platensis

This work describes the medium optimization of γ-Linolenic acid (GLA) production by Spirulina platensis using one-factor and orthogonal array design methods. In the one-factor experiments, NaHCO3 (9 mg L−1), NaNO3 (13.5 mg L−1) and MgSO4·7H2O (11.85 mg L−1) proved to be the best components for GLA production. The optimal pH for GLA production by the alga was 9.2. Based on the delta values, NaHCO3 showed the greatest effect on the GLA production of the various factors tested, followed in decreasing order by MgSO4·7H2O, NaNO3 and K2SO4. The maximum GLA yield obtained was 19.2 mgL−1 in the presence of optimum concentrations of NaHCO3 (20 g L−1), NaNO3 (3 g L−1), MgSO4·7H2O (0.5 g L−1) and K2SO4 (1.5 g L−1). Because of the slow growth rate of the algae, the practice of robust orthogonal array methods during the optimization of medium components can result in the production of an optimal biomass and a higher GLA yield for nutraceutical applications.

Microbial production of γ-linolenic acid: Submerged versus solid-state fermentations

One of the major interests of lipid biotechnology is targeted on natural manufacturing of healthy oils containing polyunsaturated fatty acids. Of them, γ-linolenic acid (C18:3 n-6; GLA) as the key intermediate in the n-6 fatty acid family is involved to maintain the proper mammalian cell functions. Insufficient supply of GLA from agricultural and animal sources resulted in ‘hunting’ for appropriate microorganisms suitable to produce this essential fatty acid in high yield. Extensive studies on oleaginous lower filamentous fungi have led to development of two basic fermentation techniques for GLA production: submerged and solid-state fermentations. Each of the processes provide specific advantages in various applications depending on the GLA product form (GLA-rich oil, whole cells, and fermented mass) and might bring new prospects to fill marketing claims in food, feed, pharmaceutical, and veterinary fields.

Effect of alternative C2 carbon sources on the growth, lipid, and γ-linolenic acid production of spirulina (Arthrospira platensis)

The influence of different organic carbon sources (glucose, ethanol, and acetic acid) at different concentrations (0.1, 0.5, and 1.0 g/L for batch and 1.0, 2.0, and 3.0 g/L for fed-batch) were studied in the mixotrophic production (using both light and carbon source) of γ-linolenic acid (GLnA) by spirulina (Arthrospira platensis). The obtained spirulina was analyzed in terms of biomass, lipid, and GLnA production. In the batch media, increasing the concentrations of glucose, ethanol, and acetic acid led to an increase in the biomass, lipid, and GLnA production. However, carbon sources at concentrations greater than 1.0 g/L in fed-batch media appeared to have no significant effects on the above parameters. It was also demonstrated that biomass, lipid, and GLnA production using ethanol and acetic acid could be as good as those achieved with the classic glucose-based culture media.

Production of γ-linolenic acid using a novel heterologous expression system in the oleaginous yeast Lipomyces kononenkoae

A novel expression system was established in the oleaginous yeast, Lipomyces kononenkoae. The expression vector pLK-rhPHG of L. kononenkoae was constructed and using the hygromycin phosphotransferase gene and green fluorescent protein gene as reporter genes. A delta 6-fatty acid desaturase gene (D6DM) from Cunninghamella echinulata MIAN6 was then expressed in this strain. The recombinant strain accumulated about 1.2% γ-linolenic acid in the total fatty acids.

Cloning and functional characterization of a fatty acid Δ6-desaturase from Oenothera biennis: Production of γ-linolenic acid by heterologous expression in Saccharomyces cerevisiae

Evening primrose Oenothera biennis is grown commercially for γ-linolenic acid (GLA) production by the fatty acid Δ6-desaturase enzymatic reaction. Based on available sequence information, the O. biennis fatty acid Δ6-desaturase gene was cloned, using rapid amplification of cDNA ends methods and reverse transcription-polymerase chain reaction. The deduced amino acid sequence showed high similarity to the plant Δ6-desaturases, which comprised the characteristics of membrane-bound desaturases, including three conserved histidine-rich motifs, hydropathy profile, and a cytochrome b5 domain at the N-terminus. Heterologous expression of the coding sequence in Saccharomyces cerevisiae resulted in the synthesis and accumulation of GLA when provided with linoleic acid substrate.

Lipid formation and γ-linolenic acid production by Mucor circinelloides and Rhizopus sp., grown on vegetable oil

The fungi strains were tested in Bioscreen automated system to select the best nutritional source. Following, shaking submserse cultures were studied in media containing sole carbon or nitrogen source. The growth of these strains improved in media containing vegetable oil, with high concentration of lipids. The high concentration of γ-linolenic acid was obtained with M. circinelloides in culture containing sesame oil.

γ-Linolenic acid production by Cunninghamella echinulata growing on complex organic nitrogen sources

Growth of two strains of Cunninghamella echinulata on various nitrogen containing raw materials (corn gluten, corn steep, whey concentrate, yeast extract and tomato waste hydrolysate) yielded important amounts of biomass containing various quantities of γ-linolenic acid (GLA) rich cellular lipids. Especially, growth on tomato waste hydrolysate (TWH) yielded 17.6 g/l of biomass containing 39.6% oil and significant quantities of GLA corresponding to 800 mg/l GLA. Mycelium-bounded proteolytic activity was detected during early growth stages on TWH and declined thereafter, increasing the concentration of assimilable nitrogen in the medium. However, addition of glucose in the medium during the stationary phase triggered the biosynthesis of reserve lipid, since an increase of the proportion of neutral lipids from 45% to 79% in total lipids was observed, while polar lipids decreased from 35% to 12% and from 20% to 9% for glycolipids plus sphingolipids and phospholipids, respectively.

Maximizing the production of γ-linolenic acid in Mortierella ramanniana var. ramanniana as a function of pH, temperature and carbon source, nitrogen source, metal ions and oil supplementation

A locally isolated (Perryvale, Alta., Canada) fungal genus, Mortierella ramanniana var. ramanniana, was evaluated as a potential industrial producer of γ-linolenic acid (GLA). Six growth variables (pH, temperature, carbon source, nitrogen source, and metal ions and oil supplementation) were systematically manipulated. The results indicated that the GLA production for this particular strain, could be maximized by using a basal growth medium consisting of 5% dextrose and 1% yeast extract, supplemented with 5 mg/L Mn 2+ with incubation at 20 °C. The lipid yield under optimum conditions was 54.2% of the total dry biomass and consisted of 84.3% unsaturated fatty acids. The yield of GLA per gram biomass represented 13.3% of the total lipid content, slightly higher than that of the typical evening primrose oil GLA yield. The study establishes this variety as an effective intermediate fungal source of GLA.

Production of γ-linolenic acid in Lotus japonicus and Vigna angularis by expression of the Δ6-fatty-acid desaturase gene isolated from Mortierella alpina

The Δ6-fatty-acid desaturase gene isolated from Mortierella alpina 1S-4 was introduced into Lotus japonicus and Vigna angularis by Agrobacterium-mediated transformation and constitutively expressed. Two transgenic L. japonicus lines accumulated γ-linolenic acid (GLA, C18:3, n-6), which was not found in the host, at levels of up to 11.34 and 9.66% of the total fatty acids in the leaves, respectively. These results indicate that the legume plant can synthesize GLA by expressing the fungus gene. The ability to synthesize GLA was also inherited by their progenies, which accumulated levels of up to 12.82 and 13.75% of the total fatty acids in the leaves, and 2.39 and 2.65% of those in the seeds. The GLA also accumulated at levels of up to 0.44 and 1.26% of the total fatty acids in the leaves and seeds, respectively, of a transgenic V. angularis line and at 0.69% in the leaves of its progeny. These findings reveal that the modification of the fatty-acid biosynthetic pathway by genetic manipulation in order to produce specific polyunsaturated fatty acids in grain legume crops is a promising technique.

Stress-induced morphological and physiological changes in γ-linolenic acid production by Mucor fragilis in batch and continuous cultures

Batch and continuous cultures conditions were studied in order to increase γ-linolenic acid production by Mucor fragilis CCMI 142, in response to the presence of ethanol. Continuous cultures were used to add ethanol pulses to steady state pellet cultures. It was demonstrated that pellet size, which allowed homogeneous fungal cultures, can be obtained by means of pH adjustment, thus enabling steady state continuous growth at 2.90±0.05. The 5 and 2% (v/v) ethanol pulses induced hyphal morphological changes with arthrospore formation. A 1% (v/v) pulse of ethanol did not immediately affect growth, but induced morphological changes, which led to autolysis at the pellet core. A 0.5% (v/v) pulse of ethanol did not affect neither the morphology nor the physiology of the microorganism to any significant extent. The 0.5 and 1% (v/v) ethanol pulses resulted in an increase in the proportion of γ-linolenic acid production up to 11%. Data from batch cultures showed a higher enhancement of ethanol, attaining 30% of γ-linolenic acid. The increase of γ-linolenic acid content observed in batch and continuous conditions appears to be a response associated with stress induced by the ethanol which seems to be of value as an industrial medium component.

Photosynthetic characterization of a mutant of Spirulina platensis

A mutant of Spirulina(Arthrospira) platensis, strain I22,obtained by mutagenesis with ethylmethanesulfonate, was partially defective inthe production of γ-linolenic acid. However, when compared with the wildform, the I22 mutant almost lost its capacity to grow at low temperatures,although at optimal temperature growth was unaffected. Measurement of themutant's photosynthetic characteristics, including O2-evolution,Pmaxand light saturation values, revealed significantly lower values than for thewild type, in contrast to the higher content of photosynthetic pigments,chlorophyll and phycocyanin. Whereas the total activity of photosynthesis oftheI22 mutant was 58% lower than that of the wild type, the PS II activity of theI22 mutant was 23% higher. On the other hand, the I22 mutant was 69% lower inPSI activity, and the growth rate of this mutant was limited at high lightintensity. These results indicated that the defect in the PS I complex of theI22 mutant may reduce its ability to utilize light to generate the energy usedin diverse biochemical processes, including fatty acid desaturation.

γ-Linolenic acid production by solid-state fermentation of Mucorales strains on cereals

Oleaginous fungi of the genus Mucorales were screened for γ-linolenic acid (GLA) production on solid substrates containing moistened cereals. Cunninghamella elegans CCF 1318 produced the highest yields of GLA when cultivated on barley. Substrate moisture and cultivation temperature proved critical for effective GLA production. Vegetable oil supplied to the cultures improved GLA production. Rotating bottles and plastic bags were used as cultivation vessels to reproduce the conditions found in rotating drums and tray bioreactors, respectively. After 11 days of cultivation at 21°C, C. elegans produced 14.2 mg of GLA per gram of dry substrate, composed of a mixture of barley, spent malt grains (SMG) and peanut oil. GLA represented 15.6% of the total fatty acids in the lipid extract.

γ-Linolenic acid production with Thamnidium elegans by solid-state fermentation on apple pomace

Apple pomace (AP) and spent malt grains (SMG) were used as the major substrate components for the production of a high-value fungal oil containing up to 11.43% biologically active γ-linolenic acid (GLA). The solid-state fermentation (SSF) process developed for GLA production was affected by substrate composition and moisture, agitation and aeration. Under optimised conditions GLA yields as high as 3.50 g/kg moist substrate were obtained from Thamnidium elegans CCF 1456, grown on a substrate consisting of a mixture of AP and SMG impregnated with peanut oil and a nutrient solution, after 8 days of incubation.