| Aurantiochytrium limacinum is a marine fungus with rich lipid especially Docosahexaenoic Acid(DHA). Docosahexaenoic acid (DHA) is an important omega-3 polyunsaturated fatty acid, which is of important physiological functions such as promoting the development and maturity of the infant brain and retina, reducing blood fat, and preventing cardiovascular disease for the elderly. Due to its easy cultivation, fast growth, and high content of DHA in cells, Aurantiochytrium limacinum has been considered to be a good strain for production of DHA.. In this paper, the nitrogen of the medium was optimized, and the polyketide synthase (PKS) gene was cloned and transgened in the A.limacinum, which will lay basis on reducement the cost of producing DHA and improvement the biomass and DHA content of A. limacinum.In the previous work of our lab, the hydrolyzed soybean meal was used to be the nitrogen source instead of the yeast extract for the fermentation of A.limacinum, which reduced the cost for producing DHA. However, the preparation of hydrolyzed soybean meal is troublesome and the hydrochloric acid could etch the apparatus.. So it is urgent to find suitable and cheap nitrogen source to meet the industrial requirements. In this paper, A. limacinum was first cultivated with corn starch powder as the nitrogen source.The cultivation results showed that the biomass and DHA content under the yeast extract medium and corn starch powder medium were 4.4 g·L-1·d-1,8.95% and 4.1 g·L-1·d-1,8.72% respectively. The biomass and DHA content are almost at the same level, however the price of corn starch powder is one-tenth of the yeast extract, so the cost of the fermentation is reduced successfully and it has significance for industrial production. On this basis, an orthogonal test was designed, including the corn starch powder, sodium glutamate and the contents of glucose as the three factors, in order to optimize the culture medium. The results showed that when the contents of glucose, corn starch powder and sodium glutamate are 120g/L,15g/L and 20g/L, the highest biomass of 5.48 g·L-1·d-1 and highest DHA content of 12.50% were achieved. This work provided a theoretical basis for the industrial fermentation of A. limacinum.Polyketide synthase (PKS) is a key enzyme in polyunsaturated fatty acids(PUFA) synthesis of A.limacinum. Genome walking was applied to clone the full length of pksl gene and 4386bp nucleotide sequence was obtained. At the same time, the promoter region containing CAAT-box and TATA-box of pks1 was cloned for the first time. The obtainment of pks1 gene sequence provides a foundation for the study of improving DHA content at the molecular level.Then pks1 sequence was compared to pks2 sequence which was cloned by our lab previously, and the sequence of 2991-4386 bp of pksl was found to have high similarityof 78% with pks2. Analyzed using BLASTp, the sequence was found to be the propylene acyl transferase enzymes domain, which has the function to make Malonyl-CoA to load into acyl carrier protein (ACP). Due to the existence in the two PKS genes, the sequence was speculated to play an important role in the synthesis of DHA. Therefore a homologous recombination plasmid p18SCPGC-1 containing the sequence was constructed and transferred into A.limacinum. The biomass and the DHA content of the transformation strain were 5.02g·L-1·d-1 and 17.11% respectively, which were 14.6% and 37.90% higher than those of the untransformed A. limacinum.In this paper, the corn starch powder was used as nitrogen source to cultivate A.limacinum successfully for the first time, which provides experimental basis for reducing the production cost and improving the level of industrial production of A.limacinum. Cloning of pksl gene and overexpression of the enzyme domain of PKS 1 lays a foundation to understand the enzyme function about DHA synthesizing and apply genetic engineering methods to cultivate the strains with high-yielding DHA. |
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