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Molecular Mechanism Of Fucoxanthin Biosynthesis Regulated By Light And Nitrogen In Mixotrophic Phaeodactylum Tricornutum

Posted on:2023-04-23Degree:DoctorType:Dissertation
Country:ChinaCandidate:R Q YangFull Text:PDF
GTID:1521307103491644Subject:Food Science and Engineering
Abstract/Summary:PDF Full Text Request
Diatoms are the most abundant single-celled photosynthetic microalgae prevalent in the ocean,using a specific fucoxanthin-chlorophyll a/c-binding protein complex(FCP)to perform important physiological function like light capture and photosynthesis.The cellular fucoxanthin has the effects of weight loss,anticancer,antioxidant,antidiabetic,and anti-Alzheimer’s,and Phaeodactylum tricornutum is considered as the best strain for fucoxanthin production.At present,the research on the production of fucoxanthin using P.tricornutum mainly focused on autotrophic condition.Although higher biomass accumulation could be achieved under mixotrophic condition,the fucoxanthin content is much lower than that under autotrophic condition.The technology of simultaneous accumulation of biomass and fucoxanthin has not been established,especially the regulatory mechanism of key factors of light and nitrogen on its biosynthesis has not been elucidated.In this study,P.tricornutum was used as a research object to systematically evaluate the environmental and nutritional factors and established the optimal mixotrophic cultivation technology based on light regulation and nitrogen supplementation to promote the simultaneous accumulation of biomass and fucoxanthin in shaking flask system.Then metabolomics,transcriptomics and multi-omics analyses were applied to elucidate the molecular regulatory mechanisms of fucoxanthin biosynthesis enhanced by arginine-urea synergy in mixotrophic P.tricornutum.The main results are listed as follows.(1)The response pattern of P.tricornutum to initial cell density,light intensity and quality was investigated,and the optimal culture conditions were screened after comprehensive evaluation.The results indicated that when cell were mixotrophic cultured with the initial cell density of 1×107 cells mL-1,light intensity of 20μmol m-2 s-1 and the mixture of red:blue(6:1)light,the highest biomass and fucoxanthin production reached 5.53 g L-1 and 68.16 mg L-1,respectively,which were 2.76 and 2.2-fold higher than that before optimization.(2)Using the above optimized light conditions,a two-stage strategy for inducing the accumulation of fucoxanthin in P.tricornutum was established.The first stage was incubated with red:blue(6:1)light at 20μmol m-2 s-1 for 6 days,and the second stage was converted to red:blue(5:1)light at 25μmol m-2 s-1 with tryptone addition for 6 days,the maximum fucoxanthin productivity(8.22 mg L-1 d-1)was achieved at second-stage,which was the highest level in P.tricornutum as reported.The qRT-PCR results revealed that expression levels of genes participated in fucoxanthin biosynthesis encoding phytoene synthase(PSY),phytoene dehydrogenase 1(PDS1),zeaxanthin epoxidase(ZEP)and fucoxanthin-chlorophyll-protein b(ZEPb)were upregulated significantly,but the genes encoding violaxanthin de-epoxidase(VDE)was down-regulated,which promoted the accumulation of fucoxanthin.(3)The preference of nitrogen sources and the correlation with fucoxanthin biosynthesis in mixotrophic P.tricornutum were systematically evaluated.The results indicated that the mixture of tryptone and urea was essential to realize the improvement of fucoxanthin biosynthesis by light regulation and nitrogen supplementation.Analyzing the utilization rate of amnio acid in mixture nitrogen sources,arginine was found to be the best preferred amino acid by mixotrophic P.tricornutum.The highest biomass concentration(3.18 g L-1),fucoxanthin content(12.17 mg g-1)and productivity(2.68 mg L-1 d-1)were achieved using 25 mmol N L-1arginine and 5 mmol N L-1 urea as nitrogen source.The targeted amino acid analysis demonstrated that arginine,ornithine,glutamine,and glutamic acid were the four amino acids with the highest proportion in free amino acid pools.Moreover,the synergistic effect of arginine-urea significantly promoted the consumption rate of arginine to accelerate urea cycle and GS-GOGAT cycle,providing more sufficient intermediate metabolites for protein synthesis.(4)Based on extensive targeted metabolome and carotenoid-targeted metabolome analysis,metabolic synthesis patten of primary metabolites and carotenoids in mixotrophic P.tricornutum upon arginine and arginine-urea synergy was deeply studied.In the early stage of culture(4th day),most primary metabolites enriched in tricarboxylic acid cycle,arginine biosynthesis and metabolism,lipid biosynthesis were upregulated under the synergistic effect of arginine and urea.In the middle and late stages of culture(8th and 12th day),the abundance of amino acids,lipids and nucleotides was continuously down-regulated,but the contents of intermediate metabolites in the fucoxanthin biosynthesis pathway were significantly up-regulated,which promoted the accumulation of fucoxanthin in cells.The time sequence analysis of primary metabolites showed that using arginine as solo nitrogen source could significantly affect the abundance of proline,lysophosphatidylcholine,aspartic acid,asparagine,and glutamic acid to resist acid stress caused by arginine consumption.Under arginine+urea medium,amounts of primary metabolites were downregulated.Only the abundance of N-monomethyl-L-arginine was significantly up-regulated,inhibiting the conversion of arginine to nitric oxide,which indicated that the blockage of arginine assimilation was one of the reasons why cells enter the stationary phase.(5)Based on combination of transcriptomics and metabolomics,a global metabolic regulatory network of fucoxanthin biosynthesis in response to arginine-urea synergy was constructed with the arginine group as the control.In the early stage of culture(4th day),the synergistic effect of arginine-urea significantly enhanced the expression of genes involved in metabolic pathways,such as the urea cycle,glycerol metabolism,aspartate and alanine system,glycolysis,tricarboxylic acid cycle and fatty acid biosynthesis,leading to the significant upregulation of proline,aspartate/asparagine,citric acid/isocitrate,eicosapentaenoic acid and lysophosphatidylcholine abundance.Moreover,the photosynthetic system Ⅰ,photosynthetic system Ⅱ and photosynthetic pigment synthesis pathways were significantly upregulated,in which the promotion of genes taking part in chlorophyll biosynthesis pathway was higher than that in fucoxanthin biosynthesis pathway.The performance of these pathways provided sufficient precursors for the active carbon and nitrogen transfer and energy exchange between organelles,meeting the needs of rapid cell growth and life activities.As the cells entered the stationary phase(8th and 12th day),most genes encoding carbon and nitrogen metabolism pathways were significantly down-regulated under the synergistic effect of arginine-urea,but some genes in the gluconeogenesis direction and fucoxanthin synthesis pathway were significantly up-regulated with upregulation of glucose-1-phosphate,zeaxanthin andβ-carotene abundances,achieving the simultaneous accumulation of biomass and fucoxanthin under the synergistic effect of arginine-urea.The trend analysis of gene expression in arginine group demonstrated that most of the significant clustering genes involved in cell growth and replication were upregulated,which explained the redivision of cells at 8th day.While the downregulation of genes encoding protein processing and photosynthesis was not conducive to the accumulation of fucoxanthin.However,most significant clustering genes in arginine+urea medium showed the trend of downward,especially genes involved in fucoxanthin biosynthesis.The results indicated that the intracellular material exchange and energy transfer were slowed down,leading to a stabilization phase of cells and inhibition of fucoxanthin accumulation.
Keywords/Search Tags:Phaeodactylum tricornutum, Fucoxanthin, Mixotrophic cultivation, Omics analysis, United-analysis
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