Remodeling of intermediate metabolism in the diatom Phaeodactylum tricornutum under nitrogen stress.

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 the gene encoding for nitrate reductase, a key enzyme required for the assimilation of nitrate. The strain exhibits 40-50% of the mRNA copy numbers, protein content, and enzymatic activity of the wild type, concomitant with a 43% increase in cellular lipid content. We suggest a negative feedback sensor that couples photosynthetic carbon fixation to lipid biosynthesis and is regulated by the nitrogen assimilation pathway. This metabolic feedback enables diatoms to rapidly respond to fluctuations in environmental nitrogen availability.

Overexpression of a diacylglycerol acyltransferase gene in Phaeodactylum tricornutum directs carbon towards lipid biosynthesis.

Under nutrient deplete conditions, diatoms accumulate between 15% to 25% of their dry weight as lipids, primarily as triacylglycerols (TAGs). As in most eukaryotes, these organisms produce TAGs via the acyl-CoA dependent Kennedy pathway. The last step in this pathway is catalyzed by diacylglycerol acyltransferase (DGAT) that acylates diacylglycerol (DAG) to produce TAG. To test our hypothesis that DGAT plays a major role in controlling the flux of carbon towards lipids, we overexpressed a specific type II DGAT gene, DGAT2D, in the model diatom Phaeodactylum tricornutum. The transformants had 50- to 100-fold higher DGAT2D mRNA levels and the abundance of the enzyme increased 30- to 50-fold. More important, these cells had a 2-fold higher total lipid content and incorporated carbon into lipids more efficiently than the wild type (WT) while growing only 15% slower at light saturation. Based on a flux analysis using 13 C as a tracer, we found that the increase in lipids was achieved via increased fluxes through pyruvate and acetyl-CoA. Our results reveal overexpression of DAGT2D increases the flux of photosynthetically fixed carbon towards lipids, and leads to a higher lipid content than exponentially grown WT cells.

An RNA interference knock-down of nitrate reductase enhances lipid biosynthesis in the diatom Phaeodactylum tricornutum.

When diatoms are stressed for inorganic nitrogen they remodel their intermediate metabolism and redirect carbon towards lipid biosynthesis. However, this response comes at a significant cost reflected in decreased photosynthetic energy conversion efficiency and growth. Here we explore a molecular genetics approach to restrict the assimilation of inorganic nitrogen by knocking down nitrate reductase (NR). The transformant strain, NR21, exhibited about 50% lower expression and activity of the enzyme but simultaneously accumulated over 40% more fatty acids. However, in contrast to nitrogen-stressed wild-type (WT) cells, which grow at about 20% of the rate of nitrogen-replete cells, growth of NR21 was only reduced by about 30%. Biophysical analyses revealed that the photosynthetic energy conversion efficiency of photosystem II was unaffected in NR21; nevertheless, the plastoquinone pool was reduced by 50% at the optimal growth irradiance while in the WT it was over 90% oxidized. Further analyses reveal a 12-fold increase in the glutamate/glutamine ratio and an increase NADPH and malonyl-CoA pool size. Transcriptomic analyses indicate that the knock down resulted in changes in the expression of genes for lipid biosynthesis, as well as the expression of specific transcription factors. Based on these observations, we hypothesize that the allocation of carbon and reductants in diatoms is controlled by a feedback mechanism between intermediate metabolites, the redox state of the plastid and the expression and binding of transcription factors related to stress responses.

Diatoms: a fossil fuel of the future.

Long-term global climate change, caused by burning petroleum and other fossil fuels, has motivated an urgent need to develop renewable, carbon-neutral, economically viable alternatives to displace petroleum using existing infrastructure. Algal feedstocks are promising candidate replacements as a 'drop-in' fuel. Here, we focus on a specific algal taxon, diatoms, to become the fossil fuel of the future. We summarize past attempts to obtain suitable diatom strains, propose future directions for their genetic manipulation, and offer biotechnological pathways to improve yield. We calculate that the yields obtained by using diatoms as a production platform are theoretically sufficient to satisfy the total oil consumption of the US, using between 3 and 5% of its land area.

Double mutation in photosystem II reaction centers and elevated CO2 grant thermotolerance to mesophilic cyanobacterium.

Photosynthetic biomass production rapidly declines in mesophilic cyanobacteria grown above their physiological temperatures largely due to the imbalance between degradation and repair of the D1 protein subunit of the heat susceptible Photosystem II reaction centers (PSIIRC). Here we show that simultaneous replacement of two conserved residues in the D1 protein of the mesophilic Synechocystis sp. PCC 6803, by the analogue residues present in the thermophilic Thermosynechococcus elongatus, enables photosynthetic growth, extensive biomass production and markedly enhanced stability and repair rate of PSIIRC for seven days even at 43 °C but only at elevated CO(2) (1%). Under the same conditions, the Synechocystis control strain initially presented very slow growth followed by a decline after 3 days. Change in the thylakoid membrane lipids, namely the saturation of the fatty acids is observed upon incubation for the different strains, but only the double mutant shows a concomitant major change of the enthalpy and entropy for the light activated Q(A)(-)→Q(B) electron transfer, rendering them similar to those of the thermophilic strain. Following these findings, computational chemistry and protein dynamics simulations we propose that the D1 double mutation increases the folding stability of the PSIIRC at elevated temperatures. This, together with the decreased impairment of D1 protein repair under increased CO(2) concentrations result in the observed photothermal tolerance of the photosynthetic machinery in the double mutant.

Molecular characterization of a cDNA encoding Cu/Zn superoxide dismutase from Deschampsia antarctica and its expression regulated by cold and UV stresses.

BACKGROUND: The Copper/Zinc superoxide dismutase (Cu/ZnSOD) gene, SOD gene, was isolated from a Deschampsia antarctica Desv. by cDNA library screening. The expression of SOD gene in the leaves of D. antarctica was determined by RT-PCR and its differential expression of gene transcripts in conditions of cold and UV radiation stresses was revealed by northern blot. FINDINGS: The molecular characterization shows that SOD cDNA is 709 bp in length, which translates an ORF of 152 amino acids that correspond to a protein of predicted molecular mass of 15 kDa. The assay shows that the expression of SOD gene increases when D. antarctica is acclimatised to 4 degrees C and exposed to UV radiation. These results indicate that the SOD gene of D. antarctica is involved in the antioxidative process triggered by oxidative stress induced by the conditions of environmental change in which they live. CONCLUSION: The present results allow us to know the characteristics of Cu/ZnSOD gene from D. antarctica and understand that its expression is regulated by cold and UV radiation.

Cloning and constitutive expression of Deschampsia antarctica Cu/Zn superoxide dismutase in Pichia pastoris.

BACKGROUND: Deschampsia antarctica shows tolerance to extreme environmental factors such as low temperature, high light intensity and an increasing UV radiation as result of the Antarctic ozone layer thinning. It is very likely that the survival of this species is due to the expression of genes that enable it to tolerate high levels of oxidative stress. On that account, we planned to clone the D. antarctica Cu/ZnSOD gene into Pichia pastoris and to characterize the heterologous protein. FINDINGS: The Copper/Zinc superoxide dismutase (Cu/ZnSOD) gene, SOD gene, was isolated from a D. antarctica by cDNA library screening. This SOD gene was cloned in the expression vector pGAPZalphaA and successfully integrated into the genome of the yeast P. pastoris SMD1168H. A constitutive expression system for the expression of the recombinant SOD protein was used. The recombinant protein was secreted into the YPD culture medium as a glycosylated protein with a 32 mg/l expression yield. The purified recombinant protein possesses a specific activity of 440 U/mg. CONCLUSION: D. antarctica Cu/ZnSOD recombinant protein was expressed in a constitutive system, and purified in a single step by means of an affinity column. The recombinant SOD was secreted to the culture medium as a glycoprotein, corresponding to approximately 13% of the total secreted protein. The recombinant protein Cu/ZnSOD maintains 60% of its activity after incubation at 40 degrees C for 30 minutes and it is stable (80% of activity) between -20 degrees C and 20 degrees C. The recombinant SOD described in this study can be used in various biotechnological applications.

The role of photochemical quenching and antioxidants in photoprotection of Deschampsia antarctica.

Deschampsia antarctica Desv. (Poaceae) is the only grass that grows in the maritime Antarctic. Constant low temperatures and episodes of high light are typical conditions during the growing season at this latitude. These factors enhance the formation of active oxygen species and may cause photoinhibition. Therefore, an efficient mechanism of energy dissipation and / or scavenging of reactive oxygen species (ROS) would contribute to survival in this harsh environment. In this paper, non-acclimated and cold-acclimated D. antarctica were subjected to high light and / or low temperature for 24 h. The contribution of non-photochemical dissipation of excitation light energy and the activities of detoxifying enzymes in the development of resistance to chilling induced photoinhibition were studied by monitoring PSII fluorescence, total soluble antioxidants, and pigments contents and measuring variations in activity of superoxide dismutase (SOD; EC 1.15.1.1), ascorbate peroxidase (APX; EC 1.11.1.11), and glutathione reductase (GR; EC 1.6.4.2). The photochemical efficiency of PSII, measured as Fv / F m, and the yield of PSII electron transport (ΦPSII) both decreased under high light and low temperatures. In contrast, photochemical quenching (qP) in both non-acclimated and cold-acclimated plants remained relatively constant (approximately 0.8) in high-light-treated plants. Unexpectedly, qP was lower (0.55) in cold-acclimated plants exposed to 4°C and low light intensity. Activity of SOD in cold-acclimated plants treated with high light at low temperature showed a sharp peak 2-4 h after the beginning of the experiment. In cold-acclimated plants APX remained high with all treatments. Activity of GR decreased in cold-acclimated plants. Compared with other plants, D. antarctica exhibited high levels of SOD and APX activity. Pigment analyses show that the xanthophyll cycle is operative in this plant. We propose that photochemical quenching and particularly the high level of antioxidants help D. antarctica to resist photoinhibitory conditions. The relatively high antioxidant capacity of D. antarctica may be a determinant for its survival in the harsh Antarctic environment.