Metabolic Regulation Of Aurantiochytrium Limacinum ATCC MYA 1381 DHA Production Based On The Synthesis And Distribution Of Cofactors

Aurantiochytrium limacinum ATCC MYA 1381 synthesizes saturated fatty acid(mainly palmitic acid)through traditional fatty acid synthase(FAS)pathway,and polyunsaturated fatty acid(docosahexenoic acid(DHA))through polyketide synthase(PKS)pathway.Because of fast growth rate,high lipid content and simple fatty acids composition,it has been considered as a potential industrial fermentation strain which could produce algae oil DHA.However,compared with fish oil DHA,the high DHA production cost restrict the market application of algae oil DHA.Therefore,it is urgent to construct high DHA producing Aurantiochytrium strains.Mastering the mechanism of regulating DHA synthesis,finding the key metabolic pathways and key enzymes to control DHA synthesis can provide operational targets for the construction of engineering strains.It should be noted that the central metabolic pathway provides cofactors AcCoA,NADPH and ATP for DHA synthesis.So understanding the mechanism of synthesis and distribution of DHA precursors will provide theoretical guidance for the selection of genetic modification targets.Our study first investigated the effects of different compounds which influenced cofactors synthesis on DHA production by Aurantiochytrium sp.Then,metabolic flux analysis based on genome metabolic network model,transcriptomics analysis and enzyme activity analysis were used to investigate the response of Aurantiochytrium sp.to the disturbance of cofactor synthesis at metabolic flux level,transcription level and enzyme level.Subsequently,the correlation between DHA,cofactor synthesis and cell response were analyzed.Finally,we investigated whether there were specific sources of NADPH for saturated fatty acid synthesis(FAS)and DHA synthesis(PKS)pathways in Aurantiochytrium sp.by isotope deuterium labeling experiment.The main results and conclusions were as follows.(1)Malate(MAL),Rotenone(ROT)and Leucine(LEU)could influence the growth rate,lipid content and DHA content of total fatty acids by regulating the supply levels of NADPH,ATP and AcCoA in Aurantiochytrium sp.respectively.And the results showed that 6 g/L MAL could increase lipid content and DHA content of total fatty acids by 26.65% and 26.13%,respectively.20 mg/L ROT could respectively increase lipid content by 26.51%,DHA content of total fatty acids by 5.22%.10 g/L LEU could decrease lipid content by 14.36%,increase DHA content of total fatty acids by 8.15%.Among the three compounds,malate got the most significant effect on promoting DHA accumulation in Aurantiochytrium sp.(2)The results of transcriptomics,enzyme activity,extracellular metabolites and metabolic flux showed that exogenous malate could increase the cytoplasmic NADPH supply by enhancing the malic enzyme(ME)pathway.At the same time,the enhancement of oxidative phosphorylation and substrate level phosphorylation pathway led to the enhancement of ATP supply.Therefore,the increase of NADPH and ATP supply were helpful to accelerate lipid synthesis rate in Aurantiochytrium sp.In addition,the results of transcriptome sequencing showed that central carbon metabolism pathway and lipid metabolism pathway were the two most significant pathways to up regulate the number and enrichment level of differentially expressed genes after adding malate.In lipid metabolism pathway,dgat,acc,fas,pksa,pksc and other lipid and fatty acid synthesis genes were up-regulated,which provided the side evidences for the improvement of lipid content and DHA content in Aurantiochytrium sp.In central carbon metabolism pathway,Aurantiochytrium sp.could keep balance of supply and demand of AcCoA and oxaloacetic acid in mitochondria by regulating the flux ratio of malate node in cytoplasm and pyruvate node in mitochondria to downstream branch pathway.And this regulation mechanism was helpful to improve the utilization efficiency of NADPH in cytoplasm.Therefore,malic enzyme gene and pyruvate carboxylase gene can be regarded as key target genes to improve DHA production in the future.(3)The deuterium labeling isotope tracing experiment results indicated that the supply sources of NADPH required by FAS pathway and PKS pathway were different.There was no significant difference in the distribution of NADPH produced by malic enzyme pathway between FAS pathway and PKS pathway.While PKS pathway preferred to use NADPH produced by pentose phosphate pathway to synthesize polyunsaturated fatty acids,such as DHA.Therefore,the activated malic enzyme pathway in Aurantiochytrium sp.provided more NADPH for the accumulation of lipid,but it will not affect the fatty acid compositions of Aurantiochytrium sp.In summary,this study screened out several modulators to enhance DHA yield by regulating the supply level of cofactors in Aurantiochytrium sp.And the metabolism regulation mechanism of malate promoting DHA synthesis in Aurantiochytrium sp.was successfully revealed through the combination of multiple analysis methods.This research will provide theoretical guidance for the optimization of fermentation conditions for Aurantiochytrium sp.and the directed genetic transformation of high DHA producing strains.