Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum

Abstract

BackgroundPhaeodactylum tricornutum is a unicellular diatom in the class Bacillariophyceae. The full genome has been sequenced (<30 Mb), and approximately 20 to 30% triacylglyceride (TAG) accumulation on a dry cell basis has been reported under different growth conditions. To elucidate P. tricornutum gene expression profiles during nutrient-deprivation and lipid-accumulation, cell cultures were grown with a nitrate to phosphate ratio of 20:1 (N:P) and whole-genome transcripts were monitored over time via RNA-sequence determination.ResultsThe specific Nile Red (NR) fluorescence (NR fluorescence per cell) increased over time; however, the increase in NR fluorescence was initiated before external nitrate was completely exhausted. Exogenous phosphate was depleted before nitrate, and these results indicated that the depletion of exogenous phosphate might be an early trigger for lipid accumulation that is magnified upon nitrate depletion. As expected, many of the genes associated with nitrate and phosphate utilization were up-expressed. The diatom-specific cyclins cyc7 and cyc10 were down-expressed during the nutrient-deplete state, and cyclin B1 was up-expressed during lipid-accumulation after growth cessation. While many of the genes associated with the C3 pathway for photosynthetic carbon reduction were not significantly altered, genes involved in a putative C4 pathway for photosynthetic carbon assimilation were up-expressed as the cells depleted nitrate, phosphate, and exogenous dissolved inorganic carbon (DIC) levels. P. tricornutum has multiple, putative carbonic anhydrases, but only two were significantly up-expressed (2-fold and 4-fold) at the last time point when exogenous DIC levels had increased after the cessation of growth. Alternative pathways that could utilize HCO3- were also suggested by the gene expression profiles (e.g., putative propionyl-CoA and methylmalonyl-CoA decarboxylases).ConclusionsThe results indicate that P. tricornutum continued carbon dioxide reduction when population growth was arrested and different carbon-concentrating mechanisms were used dependent upon exogenous DIC levels. Based upon overall low gene expression levels for fatty acid synthesis, the results also suggest that the build-up of precursors to the acetyl-CoA carboxylases may play a more significant role in TAG synthesis rather than the actual enzyme levels of acetyl-CoA carboxylases per se. The presented insights into the types and timing of cellular responses to inorganic carbon will help maximize photoautotrophic carbon flow to lipid accumulation.

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

“…Consistent with previous reports (Valenzuela et al, 2012;Mus et al, 2013;Yang et al, 2013), our results show that genes involved in lipid metabolism, glycolysis, TCA cycle, and pyruvate metabolism were upregulated significantly during TAG accumulation. Among these processes, lipid metabolism was directly Cultures were grown in f/2 (NaNO 3 concentration was reduced to 500 mM) enriched artificial seawater medium, and metabolites were analyzed at days 2, 6, and 10.…”

“…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.…”

“…Very recently, several laboratories have used RNA-seq to investigate differential gene expression during enhanced lipid production as a consequence of nitrogen depletion in P. tricornutum (Valenzuela et al, 2012;Yang et al, 2013). These transcriptomic data have revealed extensive changes in cellular transcript levels in response to nitrogen depletion and provides insights into the molecular mechanisms of lipid accumulation and candidate genes of potential importance for lipid metabolism in diatoms.…”

RNA-Seq and metabolic flux analysis of Tetraselmis sp. M8 during nitrogen starvation reveals a two-stage lipid accumulation mechanism

“…The up-regulation of genes (e.g. for TAGlipase and ACSase) within the β-oxidation pathway during starvation phase is not completely unexpected, and has previously been observed in P. tricornutum in association with changing membrane dynamics to cope with nutrient depletion (Valenzuela et al, 2012). The ECH-encoding gene however, was found to be progressively down-regulated after 24 h, and could present a potential bottleneck in the pathway.…”

“…Tetraselmis sp. Even in the absence of a fully sequenced and annotated genome, transcriptomic analysis by microarrays or RNA-Seq can provide a powerful tool to improve our understanding of the underlying physiological networks that allow microalgae to respond to environmental changes (Rismani-Yazdi et al, 2012; Valenzuela et al, 2012;Sun et al, 2013). In the present study, we gain insights into the lipid accumulation mechanism of the genus Tetraselmis, particularly the expression of genes in the FA synthesis, TAG synthesis and β-oxidation oxidation pathways, as cells transition from growth phase into stationary phase.…”