Fermentation Optimization,Molecular And Thermodynamic Mechanisms Of Squalene Production By Thraustochytrids

Squalene as the natural product with antioxidant capacity,have the functions of regulating cholesterol metabolism,resisting tumor and improving immunity.It is widely used in food,medicine,and cosmetics chemistries.The sources of plants and animals have certain limitations due to the environmental pollution and sustainability goals.The production of squalene by microorganisms has the advantages of lower cost,faster fermentation speed and controllable process,it is an inevitable trend under the carbon peaking and carbon neutrality goals,showed broad application prospects.Two thraustochytrid strains from different genus were explored in this study,one strain(Aurantiochytrium sp.TWZ-97(TWZ-97))was isolated by our group from coastal seawater of China,and another strain(Thrastochytrium sp.ATCC26185)was purchased from ATCC.The fermentation conditions were optimized for high squalene production of strain TWZ-97,and the regulation mechanism of improving squalene production by medium optimization was explored.Our result indicated that Na~+induced the increasing of squalene production.The transcriptomics,proteomics and metabolomics were used to reveal the molecular mechanism of squalene production induced by Na~+in strain ATCC26185.And we clarified the thermodynamic mechanism of Na~+regulating squalene metabolism.The specific research contents are as follows:(1)The thraustochytrid strain(TWZ-97)with high squalene production was screened from 9 strains with squalene-producing ability isolated from coastal seawater of China(Guangxi province,Fujian province and Zhejiang province).The strain TWZ-97produced squalene up to 188.60 mg/L in optimized medium(Yeast extract:25 g/L,MSG:3 g/L,glucose:40 g/L,Na Cl:6 g/L),6-fold higher than that with the initial growth conditions.(2)The effects of culture optimization on squalene metabolisms and genes expression of key enzymes were studied by transcriptomic and q PCR analysis.The results showed that optimization of culture increased genes expression of key enzymes in squalene synthesis pathway,fatty acid degradation pathway,and the amino acids metabolism related to acetyl-Co A biosynthesis,reduced the genes expression of key enzymes in fatty acid biosynthesis pathway.Transcriptomics results also provide the first experimental evidence of a complete squalene biosynthetic pathway and identified gene sequences of seven key enzymes(HMGS,HMGR,PMK,MDD,IDI,FPS and SQS).(3)The squalene production of thraustochytrids(TWZ-97 and ATCC26185)showed significant difference under different conditions with or without Na~+supplementation.Na~+induced squalene accumulation specifically.Transcriptomic analysis of strain ATCC26185 showed that Na~+increased the gene expression of key enzymes(ACAT,HMGs,HMGR,MVK,PMK and MDD)in squalene synthesis pathway,decreased the gene expression of fatty acid synthase,and promoted the gene expression of key enzyme(NDUFV1)in cell energy metabolism pathway.(4)On the basis of transcriptomics analysis,proteomics was used to further explore the molecular mechanism of squalene production enhancement induced by Na~+.Proline and glutamate are two commonly known compatible solutes for osmotic adjustment.In our proteomics analysis,OAT and P5CDH,two key enzymes catalyzing the generation of proline and glutamate,showed significantly higher levels in cells grown with sodium.And the key enzymes in MVA pathway showed higher levels,the key enzymes(HK,ALDO and PGK)in glycolysis pathway showed significantly higher abundance,and the key enzymes(ACAD)inβ-oxidation pathway showed significantly lower abundance in cells grown with sodium.These above-described results indicated the glycosis pathway flux was decreased and theβ-oxidation pathway flux was increased,the energy metabolism shift from carbohydrate to lipid oxidation.(5)The comparative metabolomic study indicated that the higher abundance of several amino acids(serine,glycine,cysteine,alanine and valine)and intermediates(3PG and pyruvate)from the EMP pathway in cells grown with sodium,These above-described results showed the increased flux of gluconeogenesis pathway and the decreased flux of EMP pathway.The result of lower fatty acid(C16:0 and C18:1)content further proved the increased flux ofβ-oxidation pathway.All the results showed cells shifted ATP generation pathway from carbohydrate metabolism to the more efficient lipid oxidation,improved the efficiency of ATP generation and promoted the biosynthesis of squalene.(6)The thermodynamic analysis of squalene biosynthesis pathway of thraustochytrid showed that the squalene biosynthesis was limited by the thermodynamics of the reaction catalyzed by ACAT in MVA pathway.The increase of squalene production induced by Na~+was not related to the content of acetyl-Co A.The results of ATP content and cell respiration rate showed the increasing of ATP consumption.Therefore,Na~+induced the energy metabolism shifting from carbohydrate metabolism to lipid oxidation,provided the strong thermodynamic driving force the thermodynamic limitation of acetyl-Co A reaction catalyzed by ACAT and promoted the biosynthesis of squalene.In conclusion,the thraustochytrid strain with high squalene production was isolated,the molecular mechanism of improving squalene production by fermentation optimization was revealed.The sodium-induced enzyme kinetics and thermodynamic mechanism of squalene production were studied from transcription,protein and metabolites levels.