| DHA (Docosahexaenoic acid) is a kind of n-3 polyunsaturated fatty acids (PUFA), which process the function of promoting the development of neural cells; tumor suppression; anti inflammatory; cardiovascular tissue protection and so on. What is more, DHA is of great importance in the development of vision and intelligence. Aurantiochytrium is a kind of unicellular marine fungi which contains rich lipid and polyunsaturated fatty acid (PUFA), especially DHA (Docosahexaenoic acid, C22:6n-3), and the lipid content reaches 50% of the dry cell. Aurantiochytrium has the advantages of safety, high quality, non-poison, easy cultivatation and so on. It draws more and more attentions in the field of human health and breed aquatics. In this paper, extraction methods of lipid and DHA, construction of DHA synthesizing related protein overexpression strains of Aurantiochytrium were investigated to improve the content of fatty acids in Aurantiochytrium cell, as well as the output of fatty acids extraction in industry.Aurantiochytrium cell contains the rich lipid, but the robust cell wall prevents the release of intracellular products. This study investigated three extraction methods-enzymolysis, ultrasound and soxhlet to disrupt the Aurantiochytrium cells and extract lipid and DHA. Several enzymes and different conditions (temperature, pH, digesting time, dosage) had been systematic studied. The results showed that the cell disruption efficiency of diffetent enzymes was papain>neutral protease> alkaline protease> cellulose> glucanase, snailase> lysozyme, and the optimized enzymatic condition was:4000U/ml of papain worked under pH6.5,45℃ for 4 hours. Then neutral protease was found to improve the lipid extraction efficiency when it was added in the enzymic system with papain. The compound enzymolysis was considered as the best condition for the lipid extraction from Aurantiochytrium. The lipid extraction efficiency (L%) of soxhlet extraction, ultrasound and optimal enzymolysis were 61.42±1.90%,58.20±3.91% and 56.37±1.41% respectively. The DHA extraction efficiency (Dw%) of enzymatic treatment, soxhlet and ultrasound were 12.82±0.66%,12.06±0.74%, and 12.02±2.09% respectively. Through enzymatic extraction, although cell disruption is weakest, yet DHA extraction efficiency is higher than another two methods. This study provides the experimental basis for the large scale extraction of DHA from Aurantiochytriwn.Acyl carrier protein (ACP) is an important protein in Aurantiochytrium cells for lipid production. In this study, acp gene was cloned. The analysis of the gene shows that it contains one ORF coding a protein of 152 amino acids (ACP). The protein is about 16.44kDa, the PI is 5.63. ACP is the intermediate carrier in the fatty acid synthesis. The active site locates on the 37th locus, a Serine residue for the pan acyl phosphate cysteamine binding. During the synthesis process, ACP is coupled with prothetic group and transports the prothetic group to the synthesizing fatty acid. Then ketone acetate acyl-ACP synthetase, ketone acetate acyl-ACP reductase, ene ester isomerase acyl-ACP dehydration and olefin ester acyl-ACP reductase will participate to synthesize PUFAs.Ketone acetate acyl acyl carrier protein reductase (KR), Dehydration isomerase (DH) is another two enzymic domains in the PUFA synthesis process which have been cloned in our laboratory. And expression vectors p18SZPC-KR-Cm, p18SZPC-DH-Cm have been constructed as well. In this study, the expression vector p18SZPC-ACP-Cm which contains acp gene was constructed, and all the 3 vectors were transformed into Aurantiochytrium cells.Then clonal strains were selected and the biological character (cellular morphologies, colony morphologies, growth rates et al.) was measured by microscopic observation and spectrochemical analysis, lipid including PUFA (high ratio of DHA) was extracted and the content and composition were tested and analyzed by GC-MS system. The result showed that the transformational strains-KR+,DH+, ACP+ had higher biomass of 19.69±2.36 g/1,19.02±2.27g/l and 18.73±0.83g/l respectively than the untransformed strain(17.81±0.90 g/1). The lipid content of the transformational strains-KR+,DH+, ACP+ were 48.07±4.31%,42.64 ±2.64% and 39.00±5.00%, which were higher than the untransformed strain(36.24 ±3.76%). While the whole DHA content of the transformational strains were KR+ strain:8.47±2.26%, ACP±strain:7.98±0.39%, DH±strain:7.19+1.75%, A.limacinum OUC 168:6.71±1.60%. The DHA concent of KR+, ACP+, DH+ strains are 26.23% ,18.93% and 7.15% higher than A.limacinum OUC 168.This study optimized enzymic disruption method and provides the experimental basis for the large scale extraction of DHA from Aurantiochytrium; construction of DHA synthesizing related protein overexpression strains may provide a basis to improve DHA content of Aurantiochytrium cell. |
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