| Long chain polyunsaturated fatty acids(LC-PUFAs) are very important to health bene?ts and are now widely accepted in the market place. There is a growing broad application value using fungal fermentation and transgenic technology in industrial production. In order to achieve a large number of high-quality LC-PUFAs, it is necessary to understand fatty acid synthesis pathway and its regulation mechanism. Up to now,few studies focus on the regulatory mechanism of desaturase gene expression.Some lower eukaryotes such as filamentous fungi can produce large amounts of PUFAs. In this work, the fermentation conditions were optimized for strain AGED and the molecular regulatory mechanism of desaturase genes have been studied. The research conclusions are as follows:The strain AGED was identificated by morphological and molecular identification and named as Mortierella sp. AGED. Influences of medium and culture conditions on biomass, lipid and ARA production were further investigated. The results showed that 5% glucose and 1% soybean meal were the best carbon and nitrogen sources for fungal growth, lipid and ARA accumulation. The best culture conditions are as follows: inoculums 8%, initial fermentation pH6, fermentation temperature 20?. The effects of precursors on the production of oil were evaluated. The results showed that 1.5% ethanol affected the yield of PUFAs significantly.? 6 desaturase gene and its 5' upstream regulatory sequence of Mortierella sp. AGED were cloned and analyzed. Results showed that 1,375 bp ? 6 desaturase gene AGfad6 contains 1,275 bp ORF encoding 424 amino acids without signal peptide. The putative protein contained three typical conserved histidine rich motifs and a typical N-terminal Cyt b5 like domain with conserved HPGG motif, with a mass of 48.3 kDa and isoelectric point of 5.96. AGfad6 gene was successfully expressed in Pichia pastoris, which exerted the effect on converting linoleic acid to ?-linolenic acid. The 1,712 bp of upstream region contained basic transcriptional elements including TATA box and GATA box, putative target-binding sites for transcription factors such as TBP(TATA binding protein), CAP(transcription activator), C/EBPalp(CCAAT-enhancer-binding protein), AP1(activator protein 1), ADR1(alcohol dehydrogenase gene regulator 1) and p40x(metabolic regulators in fungi), stress-related elements including GT-1(light-responsive, salicylic acid-inducible), STRE(stress response element), HSE(heat stress-responsive element), which might participate in regulation of PUFAs synthesis.The regulation of lipid production and desaturase genes expression in Mortierella sp. AGED were investigated in this study. Both of the fatty acid composition and ? 9-, ? 12, ? 6- and ? 5 desaturase genes mRNA levels were significantly altered under low temperature and ethanol. All desaturase genes transcription levels were increased differently. Different metal ions such as Ca2+, Zn2+ and Fe3+ could stimulate the PUFA synthesis and up-regulate desaturase genes transcription in Mortierella sp. AGED while Cu2+ inhibited desaturase genes expression and lipid accumulation. Although salicylic acid addition may facilitate the oil production, it did not promote desaturase genes expression. This indicates that fatty acid synthesis and fatty acid desaturase expression are dependent on the different external stimuli and regulated by different metabolic network.In our work, we found that desaturase genes transcription as well as lipid accumlation can be greatly affect by environmental factors such as temperature, ethanol and metal ions in Mortierella sp. AGED. The present finding could enable us to understand the regulatory mechanism of desaturase genes and provide theoretical support for future research. |
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