Metabolic Mechanism Of ω-3 Polyunsaturated Fatty Acid Synthesis In Schizochytrium Through Polyketide Synthase Pathway

Polyunsaturated fatty acids(PUFAs),especially ω-3 docosahexaenoic acid(DHA)and eicosapentaenoic acid(EPA),have been widely used in food,medical service,health care and other fields,and human’s demand for them is gradually increasing.Schizochytrium has been used as one of the main sources of DHA production,because of its large biomass,easy heterotrophic fermentation and rich lipid content.However,the mechanism of PUFAs synthesis by polyketide synthase(PKS)pathway is still unclear,and the yield of other PUFAs in Schizochytrium remains at a low level,limitting the further development of the production of PUFAs.In this work,Schizochytrium ATCC1381 was used as the starting strain for genetic modification.The effects of PKS gene clusters on the synthesis of PUFAs and their metabolic mechanisms were analyzed based on transcriptome and comparative metabonomics.Schizochytrium was then domesticated with high concentration of glucose.PUFAs yield were further improved by fed-batch fermentation with the domesticated strain.The mechanism of PUFAs synthesis by PKS pathway in Schizochytrium was finally speculated and and the metabolic regulation strategies prososed as well.The main conclusions are as follows:(1)Malonyl-CoA:ACP transacylase(MAT)was overexpressed through homologous recombination production in Schizochytrium.The results showed that lipids and PUFAs yields in MAT overexpressing strain were increased by 10.1 and 24.5%,respectively.Metabolomics analysis revealed that the intracellular tricarboxylic acid cycle was weakened and glucose absorption was accelerated in the engineered strain.In the mevalonate pathway,intracellular carotene content was decreased,and the carbon flux was then redirected towards PUFAs synthesis.Furthermore,a glucose fed-batch fermentation was finally performed with the engineered Schizochytrium.The total lipids yield was further increased to 110.5 g/L,39.6%higher than the wild strain.DHA and EPA yields were enhanced to 47.39 g/L and 1.65 g/L with an increase of 81.5 and 172.5%,respectively.(2)Disruption of chain length factor(CLF)and dehydrogenase(DH)genes were conducted in Schizochytrium.The results showed that DH and CLF-disrupted strains had significant decreases(65.85 and 84.24%)in PUFAs yield,while the saturated fatty acids(SFAs)proportion in lipids was slightly increased.Meanwhile,the disruption of CLF decreased the C-22 PUFA proportion by 57.51%without positive effect on C-20 PUFAs accumulation while DH-disrupted mutant decreased the production of each PUFA.Combined with protein prediction,it was suggested that CLF gene exerted an enormous function on the carbon chain elongation in PUFA synthesis,especially for the final elongation from C20-C22 PUFAs.Metabolomics analysis also indicated that the disruption of both genes resulted in the decrease of PUFAs but increase of SFAs,thus weakening glycolysis and tricarboxylic acid(TCA)cycle.In addition,the knockdown of ketoacyl reductase(KR)and ketoacyl synthase(KS)by CRISPR Cas9 decreased the ratio of DPA and DHA(12-16%)and increased the content of saturated fatty acids.Based on the results,it was speculated that MAT,ER and DH genes played a role in the synthesis of lipidsin both PKS and FAS patRanelletti hways.KR,KS and CLF are essential in PKS pathway.(3)Strain domestication with high concentration of glucose was done in this study.Compared with the wild strain,the total lipid content in Schizochytrium sp.HG-20 was increased by 54.38%,reaching 27.4 g/L,and the DHA and EPA ratio in the lipids were increased by 18.12 and 204.14%,respectively.Metabolomics and transcriptomics analysis were conducted.Compared with the wild strain,the domesticated strains had a significant improvement in glycolysis,FAS and PKS pathway metabolism,in which the key genes GAPD,FabG1 and KR were the highest up-regulated genes.The amino acid synthesis and pentose phosphate pathway(PPP),as the competitive pathways,were decreased,which further increased the PUFAs synthesis.Moreover,the fed-batch fermentation was carried out with the domesticated strains.Compared with the wild strain,the biomass and lipid content of the domesticated strain were increased by 75.2 and 15.5%,respectively,reaching 241.3 g/L and 94.6 g/L.DHA and EPA content were increased by 23.77 and 124.70%,reaching 34.89 g/L and 1.74 g/L yield,respectively.(4)Four kinds of benzoic acid derivatives promoted the production of lipids by Schizochytrium.The yield of lipids was increased by 56.84%in the comparision of control group with aminobenzoic acid(ABA)at a concentration of 200 mg/L.The metabolomics analysis showed that 200 mg/L ABA had the optimal effect on TCA cycle,glycolysis,mevalonate pathway and lipid synthesis.Moreover,the addition of p-aminobenzoic acid could promote the synthesis of tetrahydrofolate,enhance the downstream of metabolic process in PPP,and then increase NADPH,and ultimately promote the flow of carbon to the lipids synthesis by PKS and FAS.