Introduction: Arachidonic acid (5, 8, 11, 14-eicosatetraenoic acid, ARA), a long chain polyunsaturated fatty acid (PUFA) of the n-6 class, plays an important roles in the structure and function of biological membranes. ARA has also attracted attention as a precursor of prostaglandins, thromboxane, prostacyclin, and leucotrienes, which have potent and various physiological actions including uterine muscle contraction, relaxation, vasodilatation, and antihypertensive action. Lower fungi of Zygomycetes especially Mortierella alpina were served as rich sources of ARA. In this research the time courses for biomass, lipid and arachidonic acid production by Mortierella alpina CBS 754.68 with different levels of glucose and soybean concentrations were examined. Afterward the effect of substrates levels on expression of the elangase genes involved in biosynthesis of ARA was studied in a shaker flask scale.
Materials and methods: Mortierella alpina CBS 754.68 was purchased from Centraal Bureau Schimmel cultures (CBS, the Netherlands). The seed culture medium contained (g/L): glucose 30 and yeast extract 7. The seed culture of 100 mL was incubated at 25 °C for two days in a gyratory shaker at 185 rpm. The fermentation medium was inoculated of with 5% (v/v) of the mycelium suspension of the seed culture. Fermentation was carried out at 21 °C; pH 6 for ten days with glucose (70 and 50 g/L) and soy bean meal (20 and 10 g/L) as carbon and nitrogen resources, respectively. The dinitrosalicylic acid (DNS) method was used to determine the reducing sugar concentration. Protein concentration was assayed using the Lowry’s method. Total RNA was extracted using the RNA Kit according to the manufacturer’s instructions (Invitrogen) The NCBI database was scanned for genes encoding for elongases. The hints were aligned and the conserved regions were used for primer designing. PCR primers for mentioned genes and the Housekeeping Gene (actin) were designed using the Gene Runner Design software. cDNA was synthesized using the cDNA synthesis kit (Invitrogen) using oligo (dT) as primers according to the manufacturer’s instructions. Expression analysis of genes involving in ARA biosynthesis was carried out using Real-time PCR. The Real-time PCR mixtures containing cDNA and each primer were heated at 95 °C for 15 min and then subjected to 40 cycles consisting of denaturation at 95 °C for 5 s, annealing at 60 °C for 20s, and extension at 72 °C for 20 s, and finally for an extension of 10 min at 72 °C. The expression levels of target genes were normalized based on actin encoding gene (ΔCt = Ct target gene − Ct reference gene). Since the total number of cycles in the Real-Time PCR was 40, for easier interpretation, the ΔCt was converted into 40-ΔCt.
Results and Discussion: Biomass growth was declined with increasing carbon content in the early days of cultivation and subsequently increased. Growth has great effect on reduction of the amount of glucose in the medium; thereby osmotic pressure had been reduced and biomass production was boosted. Protein content of media had a significant impact on biomass production. The results indicate that the protein was depleted in 2 day of fermentation. In all three media the destruction of biomass was started when the carbon source was reached the lowest level. In media with high content of protein, oil accumulation was lower than the two other media, which indicated the negative impact of protein on the lipid accumulation. In this media although the protein source along with the other medium to be depleted on day 2, In following fermentation, the biomass growth and lipid accumulation was higher and lower respectively than other medium. High concentrations of glucose had inhibitory effect on oil production while had positive effect on biomass production. Results indicated that oil accumulation significantly increased until day 6 and then the trend was slow. Reduced glucose medium can have a significant effect in this phenomenon. Initial concentrations of glucose (50 g/l) and soybean (10 g/l) have good effect on lipid production (47% of biomasss) while in this condition; the production of arachidonic acid was lower than the other media. ARA content of lipid was increased during the whole fermentation process in all treatment. It was observed that an improvement in arachidonic acid (56% of lipid) in lipid was achieved at lowest and highest levels of glucose (50 g/l) and soy bean (20 g/l) respectively. The results of this study showed that the expression of our target genes depends on culture conditions. Expression of MALC1 was influenced by culture condition. Low and high levels of carbon and nitrogen content respectively also biomass growth had great impact on MALC1 expression. A significant decrease of biomass from 6 to 8 had a significant impact in reducing MALC1 expression. the expression of GLELO gene was lowest at day 6 and increased again at day 8.
Conclusion: The results showed that the expression of GLELO gene was decreased coincided with reduced levels of carbon content less than 5 grams per liter on the sixth day. With comparison of expression of MALC1and GLELO encoding genes in optimal media for ARA production can be concluded that the GLELO gene is a rate-limiting step in the ARA biosynthesis. |
