Study On Related Genes Of Polyunsaturated Fatty Acid Synthesis Regulating Metabolism In Schizochytrium

Polyunsaturated fatty acids(PUFAs)can regulate blood lipid,protect eyesight,prevent cardiovascular diseases,and fight inflammation and allergy.Schizochytrium has been widely used in the PUFAs especially DHA production,due to high lipid content,high percentage of PUFAs and high density culture.However,the mechanism of PUFAs synthesis,especially polyketide synthase(PKS)pathway,is still unclear in Schizochytrium,and the functions of multiple catalytic domains in PKS gene cluster are still unclear.The PUFAs yield especially the EPA yield,needs to be further improved.In our study,Schizochytrium limacinum SR21(S.limacinum SR21)was selected as the starting strain.The catalytic function of enylreductase(ER)domain was investigated by gene knock-out modification.Based on the function of ER domain,the effects of exogenous addition and gene knock-in on PUFAs synthesis were investigated.In addition,the function of Elo1 elongase was investigated by heterologous expression and gene knock-out.The main results are as follows:(1)The OrfB-ER and OrfC-ER genes were knocked out by homologous recombination in S.limacinum SR21.The results showed that PUFAs content decreased significantly when OrfB-ER gene was knocked out,and saturated fatty acid(SFAs)content decreased when OrfC-ER gene was knocked out,which indicated OrfB-ER domain played a key role on PUFAs synthesis and OrfC-ER domain was related to SFAs synthesis.Based on the function of ER domain,triclosan as ER regulator was selected to regulate fatty acid synthesis.The results showed that the yields of PUFAs,EPA and DHA increased by 51.7%,53.4%and 49.3%respectively,when 8 ?M triclosan was added at 24 h.GC-MS analysis showed that the addition of triclosan enhanced glycolysis pathway,weakened TCA cycle and mevalonate pathway and promoted carbon metabolism flow to lipid synthesis.(2)The ER gene of Shewanella SCRC2738(sh-ER)was knocked into OrfB-ER and OrfC-ER gene in S.limacinum SR21 by homologous recombination,respectively.The results showed that PUFAs synthesis of C-sh-ER konck-in strain was unchanged basically,while EPA content of B-sh-ER konck-in strain was increased by 85.7%.The B-sh-ER konck-in strain was cultured in 5 L bioreactor.The results showed that the lipid content increased by 40.9%to 80.2g/L and EPA production increased by 71.6%to 732.4 mg/L.(3)A segment of Elo1 elongase gene was analyzed and identified.The amino acid sequence alignment showed that Elo1 elongase displayed 67%amino acid sequence identity with ?5 elongase of Aurantiochytrium sp.FCC1311.The function of Elo1 elongase and its effect on PUFAs synthesis were investigated by heterologous expression and gene knock-out modification.The results found that the transformed strain could extend arachidonic acid(ARA)to eicosaenoic acid(DTA)and the ARA content increased by 84.0%in Elo1 elongase gene knock-out strain.The above results indicated that the Elo1 elongase had similar functions to ?5 elongase.This study provides a new perspective for analyzing the PKS pathway synthesis mechanism and a new direction and strategy for regulating PUFAs synthesis of Schizochytrium.