Triacylglycerol accumulation and profiling in the model diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum (Baccilariophyceae) during starvation

Abstract

Although substantial economic barriers exist, marine diatoms such as Thalassiosira pseudonana and Phaeodactylum tricornutum hold promise as feedstock for biodiesel because of their ability to manufacture and store triacylglycerols (TAGs). The recent sequencing of these two marine diatom genomes by the United States Department of Energy Joint Genome Institute and the development of improved systems for genetic manipulation should allow a more systematic approach to understanding and maximizing TAG production. However, in order to best utilize these genomes and genetic tools, we must first gain a deeper understanding of the nutrient-mediated regulation of TAG anabolism. By determining both the yield and molecular species distribution of TAGs we will, in the future, be able to fully characterize the effects of genetic manipulation. Here, we lay the groundwork for understanding TAG production in T. pseudonana and P. tricornutum, as a function of nitrate and silicate depletion. Diatoms were starved of either nitrate or silicate, and TAGs were extracted with hexane from lyophilized samples taken at various time intervals following starvation. The timing of TAG production and the relative abundance of TAGs were estimated by fluorescence spectroscopy using Nile red and the total yield per biomass determined by gravimetric assay. TAGs were analyzed using thin layer chromatography, gas chromatography-mass spectrometry, and electrospray ionization mass spectrometry to identify the major TAG species produced during the growth curve. Under our conditions, the TAG yield from T. pseudonana is about 14-18% of total dry weight. The TAG yield from P. tricornutum is about 14% of total dry weight. Silicate-starved T. pseudonana accumulated an average of 24% more TAGs than those starved for nitrate; however, the chemotypes of the TAGs produced were generally similar regardless of the starvation condition employed.

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom

“…Differences in accumulation of FAME were significant for the two genotypes through 60 h as determined by a two-way ANOVA (Supplemental File 1). FAME composition in both wild-type and NR-KO14 cells was dominated by C16:0, C16:1, and C20:5 FAs (Supplemental Figures 7A to 7E), consistent with previous reports for diatom FAME composition (Abida et al, 2015;Shen et al, 2016;Yu et al, 2009). As previous studies have established (Bender et al, 2014;Hockin et al 2012;Li et al, 2014;Rismani-Yazdi et al, 2012;Shifrin and Chisholm, 1981;Yang et al 2013;Yoon et al, 2012), N-limited conditions in photosynthetic microalgae induce accumulation of lipids, predominantly TAG, which is stored in lipid droplets.…”

“…In turn, it provides the reduced amino groups that are necessary for synthesis of proteins (particularly Rubisco), the light-harvesting complexes, and photosynthetic pigments (Nunes-Nesi et al, 2010;Turpin, 1991). In NO 3 2 -limited conditions, diatoms and certain green algae redirect C from storage carbohydrates such as chrysolaminarin and starch to accumulation of neutral lipids, specifically triacylglycerol (TAG) (Rismani-Yazdi et al, 2012;Shifrin and Chisholm, 1981;Yu et al, 2009). Much recent work has focused both on characterizing the genes and metabolic pathways that induce lipid biosynthesis and on identifying routes and regulators that could be harnessed to substantially increase TAG production without negatively affecting growth (Alipanah et al, 2015;Ge et al, 2014;Hildebrand et al, 2012;Levitan et al, 2015;Radakovits et al, 2010;Trentacoste et al, 2013;Yang et al, 2013;Yoon et al, 2012).…”

Methylcrotonyl-CoA Carboxylase Regulates Triacylglycerol Accumulation in the Model Diatom Phaeodactylum tricornutum

“…Diatoms can also rapidly induce TAGs under Si limitation, which avoids the detrimental effects on photosynthesis associated with other nutrient limitations (Roessler, 1988;Yu et al, 2009). In P. tricornutum, it has been well documented that the increase in TAGs becomes noticeable as a result of nutrient stress (Larson and Rees, 1996;Yu et al, 2009;Burrows et al, 2012;Valenzuela et al, 2012Valenzuela et al, , 2013Mus et al, 2013), especially nitrogen deficiency. In our study, TAG accumulation was initiated after nitrogen was nearly depleted and the lower initial nitrate concentration resulted in an earlier TAG accumulation in cells.…”

Scenedesmus sp. NJ-1 isolated from Antarctica: a suitable renewable lipid source for biodiesel production

“…Standards (monostearin, distearin and triolein) were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Lipids were visualized by exposing the plates to iodine vapor (Yu et al 2009). BODIPY 505/515 (Invitrogen, Carlsbad, CA, USA) serving as an excellent stain for lipid bodies of live algal cells was used in this study.…”