Regulatory branch points affecting protein and lipid biosynthesis in the diatom Phaeodactylum tricornutum (original) (raw)
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Remodeling of intermediate metabolism in the diatom Phaeodactylum tricornutum under nitrogen stress
Proceedings of the National Academy of Sciences of the United States of America, 2015
Diatoms are unicellular algae that accumulate significant amounts of triacylglycerols as storage lipids when their growth is limited by nutrients. Using biochemical, physiological, bioinformatics, and reverse genetic approaches, we analyzed how the flux of carbon into lipids is influenced by nitrogen stress in a model diatom, Phaeodactylum tricornutum. Our results reveal that the accumulation of lipids is a consequence of remodeling of intermediate metabolism, especially reactions in the tricarboxylic acid and the urea cycles. Specifically, approximately one-half of the cellular proteins are cannibalized; whereas the nitrogen is scavenged by the urea and glutamine synthetase/glutamine 2-oxoglutarate aminotransferase pathways and redirected to the de novo synthesis of nitrogen assimilation machinery, simultaneously, the photobiological flux of carbon and reductants is used to synthesize lipids. To further examine how nitrogen stress triggers the remodeling process, we knocked down th...
BioEnergy Research, 2012
One approach to achieve continuous overproduction of lipids in microalgal "cell factories" relies upon depletion or removal of nutrients that act as competing electron sinks (e.g., nitrate and sulfate). However, this strategy can only be effective for bioenergy applications if lipid is synthesized primarily de novo (from CO 2 fixation) rather than from the breakdown and interconversion of essential cellular components. In the marine diatom, Phaeodactylum tricornutum, it was determined, using 13 C-bicarbonate, that cell growth in nitrate (NO 3 − )-deprived cultures resulted predominantly in de novo lipid synthesis (60 % over 3 days), and this new lipid consisted primarily of triacylglycerides (TAGs). Nearly complete preservation of 12 C occurred in all previously existing TAGs in NO 3 − -deprived cultures and thus, further TAG accumulation would not be expected from inhibition of TAG lipolysis. In contrast, both high turnover and depletion of membrane lipids, phosphatidylcholines (PCs), were observed in NO 3 − -deprived cultures (both the headgroups and fatty acid chains), while less turnover was observed in NO 3 − replete cultures. Liquid chromatographytandem mass spectrometry mass spectra and 13 C labeling patterns of PC headgroups provided insight into lipid synthesis in marine diatoms, including suggestion of an internal pool of glycine betaine that feeds choline synthesis. It was also observed that 16C fatty acid chains incorporated into TAGs and PCs contained an average of 14 13 C carbons, indicating substantial incorporation of 13 C-bicarbonate into fatty acid chains under both nutrient states. Keywords Algae . Biodiesel . Nitrate . Nutrients . Fatty acid metabolism . De novo lipid biosynthesis . Phaeodactylum tricornutum Electronic supplementary material The online version of this article (
Algal Research, 2018
Nitrogen deprivation increases the triacylglycerol (TAG) content in microalgae but also severely decreases the growth rate. Most approaches that attempted to increase TAG productivity by overexpression or knockdown of specific genes related to the regulation of the lipid synthesis have reported only little success. More insight into the molecular mechanisms related to lipid accumulation and impaired growth rate is needed to find targets for improving TAG productivity. By using the emerging "omics" approach, we comprehensively profiled the physiology, transcriptome, proteome and metabolome of the diatom Phaeodactylum tricornutum during steady state growth at both nitrogen limited and replete levels during light:dark cycles. Under nitrogen limited conditions, 22% (2699) of the total identified transcripts, 17% (543) of the proteins and 44% (345) of the metabolites were significantly differentially regulated compared to nitrogen replete growth conditions. Although nitrogen limitation was responsible for the majority of significant differential transcript, protein and metabolite accumulation, we also observed differential expression over a diurnal cycle. Nitrogen limitation mainly induced an upregulation of nitrogen fixation, central carbon metabolism and TCA cycle, while photosynthetic and ribosomal protein synthesis are mainly downregulated. Regulation of the lipid metabolism and the expression of predicted proteins involved in lipid processes suggest that lipid rearrangements may substantially contribute to TAG distribution. However, TAG synthesis is also limited by the reduced carbon flux through central metabolism. Future strain improvements should therefore focus on understanding and improving the carbon flux through central carbon metabolism, selectivity and activity of DGAT isoforms and lipase enzymes.
Whole-cell response to nitrogen deprivation in the diatom Phaeodactylum tricornutum
Algal growth is strongly affected by nitrogen (N) availability. Diatoms, an ecologically important group of unicellular algae, have evolved several acclimation mechanisms to cope with N deprivation. In this study, we integrated physiological data with transcriptional and metabolite data to reveal molecular and metabolic modifications in N-deprived conditions in the marine diatom Phaeodactylum tricornutum. Physiological and metabolite measurements indicated that the photosynthetic capacity and chlorophyll content of the cells decreased, while neutral lipids increased in N-deprived cultures. Global gene expression analysis showed that P. tricornutum responded to N deprivation through an increase in N transport, assimilation, and utilization of organic N resources. Following N deprivation, reduced biosynthesis and increased recycling of N compounds like amino acids, proteins, and nucleic acids was observed at the transcript level. The majority of the genes associated with photosynthesis and chlorophyll biosynthesis were also repressed. Carbon metabolism was restructured through downregulation of the Calvin cycle and chrysolaminarin bio-synthesis, and coordinated upregulation of glycolysis, the tricarboxylic acid cycle, and pyruvate metabolism, leading to funnelling of carbon sources to lipid metabolism. Finally, reallocation of membrane lipids and induction of de novo triacylglycerol biosynthesis directed cells to accumulation of neutral lipids.
Journal of Oceanology and Limnology, 2018
A freshwater green microalgae Chlorella sp., UMACC344 was shown to produce high lipid content and has the potential to be used as feedstock for biofuel production. In this study, photosynthetic effi ciency, biochemical profi les and non-targeted metabolic profi ling were studied to compare between the nitrogen-replete and deplete conditions. Slowed growth, change in photosynthetic pigments and lowered photosynthetic effi ciency were observed in response to nitrogen deprivation. Biochemical profi les of the cultures showed an increased level of carbohydrate, lipids and total fatty acids, while the total soluble protein content was lowered. A trend of fatty acid saturation was observed in the nitrogen-deplete culture with an increase in the level of saturated fatty acids especially C16:0 and C18:0, accompanied by a decrease in proportions of monounsaturated and polyunsaturated fatty acids. Fifty-nine metabolites, including amino acids, lipids, phytochemical compounds, vitamins and cofactors were signifi cantly dysregulated and annotated in this study. Pathway mapping analysis revealed a rewiring of metabolic pathways in the cells, particularly purine, carotenoid, nicotinate and nicotinamide, and amino acid metabolisms. Within the treatment period of nitrogen deprivation, the key processes involved were reshuffl ing of nitrogen from proteins and photosynthetic machinery, together with carbon repartitioning in carbohydrates and lipids.
South African Journal of Botany, 1989
Nitrogen and carbon metabolism of Monoraphidium fa/Galus were shown to compete for carbon skeletons under high PPFD and atmospheric levels of CO2. The uptake of N03-was dependent upon the presence of a Cj-source and was inhibited in the presence of MSX with no detectable efflux of NH4 + from the cells. These results indicate that either Cj or an intermediate of carbon metabolism was essential for the maintenance and stabilization of the N03-uptake system. The addition of inorganic-N (N03-or NH4 +) to the N-limited cells resulted in a suppression in the rates of nett photosynthetic CO2 fixation and O2 evolution. The inhibition of CO 2 fixation and O2 evolution by N03-and NH4 + was partially alleviated in the presence of MSX. The carbon skeleton demand of amino acid biosynthesis may be met by anaplerotic reactions which would drain off triosephosphates from the RPP pathway via the TCA cycle resulting in reduced rates of RuBP regeneration and increased mitochondrial respiratory activity. Stikstof-en koolstof-metabolisme van Monoraphidium fa/Galus het, onder die invloed van hoe ligintensiteite en atmosferiese CO2-vlakke, geblyk om te kompeteer vir koolstofskelette. Die opname van N03-was afhanklik van die aanwesigheid van 'n Cj-bron en was verminder in die aanwesigheid van MSX met geen waarneembare NH4 + uitvloeiing vanuit die selle. Hierdie waarnemings dui aan dat 6f Cj 6f 'n intermedier van C-metabolisme noodsaaklik was vir die instandhouding en stabilisering van die N03-opgaarsisteem. Die toevoeging van anorganiese-N (N03-of NH4 +) tot die N-beperkte selle het gelei tot die onderdrukking van netto fotosintetiese C-fiksasie en 02-evolusie. Hierdie onderdrukking van CO2-fiksasie en Orevolusie was gedeeltelik verlig in die aanwesigheid van MSX. Die behoefte vir 'n koolstofskelet in aminosuur-biosintese mag deur anaplerotiese reaksies voorsien word wat triose-fosfate, langs die weg van die TCA-siklus, uit die RPPsiklus sou dreineer, en lei tot 'n verminderde tempo van RuBP-regenerasie en toename in die respiratoriese aktiwiteit van mitokondria.
PLANT PHYSIOLOGY, 2012
The availability of nitrogen varies greatly in the ocean and limits primary productivity over large areas. Diatoms, a group of phytoplankton that are responsible for about 20% of global carbon fixation, respond rapidly to influxes of nitrate and are highly successful in upwelling regions. Although recent diatom genome projects have highlighted clues to the success of this group, very little is known about their adaptive response to changing environmental conditions. Here, we compare the proteome of the marine diatom Thalassiosira pseudonana (CCMP 1335) at the onset of nitrogen starvation with that of nitrogen-replete cells using two-dimensional gel electrophoresis. In total, 3,310 protein spots were distinguishable, and we identified 42 proteins increasing and 23 decreasing in abundance (greater than 1.5-fold change; P < 0.005). Proteins involved in the metabolism of nitrogen, amino acids, proteins, and carbohydrates, photosynthesis, and chlorophyll biosynthesis were represented....
Journal of Experimental Marine Biology and Ecology, 2010
Information on interaction of C and N at the cellular level is lacking for ecologically relevant phytoplankton species. We examined the effects of NO 3 availability on C and N fluxes in the widely distributed marine coccolithophore Emiliania huxleyi. Cells were cultured at replete (∼280 μM) and ambient (∼10 μM) NO 3 -, the latter representing a typical surface water nitrate concentration of the North Atlantic Ocean during spring. While growth rates and C to N ratios were not altered by the NO 3 availability, organic C and N as well as inorganic C quotas were reduced under ambient NO 3 -. Growth at ambient NO 3 caused a higher proportion of fixed C to be allocated to lipids relative to carbohydrates and especially to proteins. Ambient NO 3 --grown cells showed lower V max of nitrate reductase (NR) and nitrite reductase (NiR) (ambient/replete: V max NR = 0.64/1.09 fmol min -1 cell -1 ;