Formula Optimization About Artificial Seawater And Preliminary Function Research On Pks3Gene Of Schizochytrium

Docosahexaenoic acid （DHA） as an omega-3polyunsaturated fatty acid（omega-3PUFA） is widely used because of it’s important physiological functionssuch as cancer resistanting, immunity enhancing, intelligence increase, eyesightprotection and so on. Schizochytrium is a kind of marine fungi with rich DHA, whichis becoming the new resource for production of DHA because it is safe, easy tocultivate and proliferate and it contains high content and good quality of lipid. Atpresent, in Japan, the United States and some European countries, Schizochytrium hasbeen applied to industrialized production of DHA. However, in domestic, thetechnology for DHA production is still in its infancy, and low DHA yield is an urgentproblem to be resolved. Therefore, improvement of DHA production ofSchizochytrium by using various technology and biological methods is particularlyimportant. This paper carried out the following work on two aspects: one isoptimization of ion concentration in the culture medium to improve the DHAproduction of Schizochytrium, the other is cloning and study of the function of DHAsynthesis related genes of Schizochytrium.1, On the basis of substitution of the natural seawater using artificial seawater in themedium of Schizochytrium, this study tried to improve the growth and DHA contentof Schizochytrium OUC007by reducing NaCl concentration, and exploratory addingKH₂PO₄and FeSO₄·7H₂O in the artificial seawater. Results show that, properlyreduction the content of NaCl and addition KH₂PO₄and FeSO₄·7H₂O in the artificialseawater can increase the growth and DHA content of Schizochytrium OUC007tosome extent, while high concentration of KH₂PO₄and FeSO₄·7H₂O would inhibitSchizochytrium OUC007. The optimal concentration of the three kinds of salts forincreasing biomass and DHA accumulation of Schizochytrium OUC007should be: NaCl，200mmol/L；KH₂PO₄，10mmol/L；FeSO₄·7H₂O，2mmol/L. Then anorthogonal experiment of the three factors at three levels was designed and theoptimal combination of NaCl, KH₂PO₄and FeSO₄·7H₂O in the artificial seawater waspresented as: NaCl，200mmol/L；KH₂PO₄，15mmol/L；FeSO₄·7H₂O，3mmol/L.Optimized artificial seawater significantly promoted the growth and DHA content ofSchizochytrium OUC007. Under the optimal medium, the biomass and DHA contentof Schizochytrium OUC007reached6.93g/l.d and19.66%, improved48.08%and53.83%than biomass （4.68g/l.d） and DHA content（12.78%） of the control group innatural seawater; and improved39.44%and47.27%than biomass （4.97g/l.d） andDHA content （13.35%） of the control group in unoptimized artificial seawater.2, In this study, with the reported pks3genes in GenBank for reference weobtained pks3gene of Schizochytrium by PCR amplification and genome walking.Then the amino acid sequence was deduced and analyzed, and the higher structure ofthe deduced protein was forecasted. Results show that the pks3gene ofSchizochytrium consists only one exon, and the length of the open reading frame（ORF） is3697bp. The gene encodes1230amino acids, which is81%identity withpks3reported in GenBank. In addition, the amino acid sequence deduced from pks3gene was predicted to contain two conservative active domains:3-keto acyl-ACPreductase （KR） domain and dehydration/isomerase （DH） domain.To research the function of pks3gene, two knockout vectors were constructed byusing the two enzyme domains as the homologous sites and using Zeocin resistancegene-Ble as the resistant selective marker. As the result, after electric transformationof the homologous recombination vectors, two Schizochytrium strains with pks3genedeletion are screened and identified. They are transformant of KR-with KR domainabsent and transformant of DH-with DH domain absent. Both of the transformantsare significantly different from OUC168in colony morphology and size of the cell.But above all, they are slow in growth, low in biomass, and with no DHAaccumulation. It is speculated to due to the deletion of pks3gene of Schizochytrium,which not only terminates the DHA accumulation, but also breaks the cell’s metabolicbalance. Then the normal physiological activities of Schizochytrium are destroyed, leading to the growth retardation and biomass reduction of Schizochytrium.In this paper, the optimization of the artificial seawater brings a new method toimprove the biomass and DHA accumulation of Schizochytrium. The cloning andfunction study of pks3gene of Schizochytrium establishes a foundation for revealingthe synthetic pathway of DHA and function of enzymes that related to DHA synthesis.