Center for Biofilm Engineering, Bozeman, USA.
Biotechnol Biofuels. 2012 Jun 6;5(1):40. doi: 10.1186/1754-6834-5-40.
Phaeodactylum 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.
The 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).
The 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.
菱形藻是颗石藻目硅藻纲的单细胞硅藻。其全基因组序列已测序(<30Mb)，在不同的生长条件下，基础干重的三酰基甘油(TAG)积累约为 20%至 30%。为了阐明菱形藻在营养剥夺和脂质积累过程中的基因表达谱，将细胞在硝酸盐与磷酸盐比例为 20:1 (N:P)的条件下培养，并通过 RNA-seq 测定随时间监测全基因组转录物。
尼罗红（NR）荧光（每细胞的 NR 荧光）随时间增加；然而，NR 荧光的增加先于外部硝酸盐完全耗尽。外源性磷酸盐先于硝酸盐耗尽，这些结果表明，外源性磷酸盐的耗尽可能是脂质积累的早期触发因素，而硝酸盐耗尽则放大了这种积累。正如预期的那样，许多与硝酸盐和磷酸盐利用相关的基因被上调表达。在营养缺乏状态下，硅藻特异性细胞周期蛋白 cyc7 和 cyc10 下调表达，而细胞周期蛋白 B1 在生长停止后的脂质积累过程中上调表达。虽然与光合作用碳还原的 C3 途径相关的许多基因没有显著改变，但与光合作用碳同化的假定 C4 途径相关的基因在细胞耗尽硝酸盐、磷酸盐和外源性溶解无机碳 (DIC)水平时上调表达。菱形藻有多个假定的碳酸酐酶，但只有两个在生长停止后 DIC 水平增加的最后一个时间点显著上调(2 倍和 4 倍)。基因表达谱还暗示了可能利用 HCO3-的替代途径(例如，假定的丙酰辅酶 A 和甲基丙二酰辅酶 A 脱羧酶)。
研究结果表明，当种群生长停滞时，菱形藻继续进行二氧化碳还原，并且根据外源性 DIC 水平，使用了不同的碳浓缩机制。基于脂肪酸合成的整体低基因表达水平，结果还表明，乙酰辅酶 A 羧化酶前体的积累可能在 TAG 合成中发挥更重要的作用，而不是乙酰辅酶 A 羧化酶本身的实际酶水平。本研究深入了解了细胞对无机碳的反应类型和时间，将有助于最大限度地将光合作用碳流转化为脂质积累。
