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Abstract Background Nitrogen (N) starvation in algae induces a variety of structural and metabolic changes including accumulation of triacylglycerol (TAG). Given the promising prospect of using algae as feedstock for biofuel production, accumulation of TAG upon N starvation becomes an ideal system to study TAG biosynthesis. Under nitrogen-depleted conditions, algae also accumulate compatible solutes such as sugar and certain amino acids, which is expected to elevate osmolarity in the cytoplasm. However, how osmoregulation is maintained and how it impacts on carbon metabolism, especially TAG accumulation under N starvation, are not well understood. Results We show here that potassium channel KCN11 localized in the contractile vacuole (CV) mediates osmoregulation during N starvation and loss of KCN11 profoundly affects cell physiology and TAG biosynthesis. KCN11 level is increased and the CV pulsation is accelerated. Loss of KCN11 induces aberrant CV cycle, inhibition of cell growth, increase of cell size, inhibition of chlorophyll loss and TAG accumulation. These effects are rescued by addition of sucrose to raise osmolarity in the culture medium, indicating that osmoregulation is required for cell adaptation to N starvation. Metabolomic analysis shows reduction of acetyl-CoA and accumulation of glyceraldehyde-3-phosphate in kcn11 mutant relative to the control under N starvation, indicating that defects in acetyl-CoA biosynthesis and some metabolic steps from glyceraldehyde-3-phosphate to TAG contribute to the decreased TAG accumulation due to loss of osmoregulation. Conclusions This work provides novel insight of osmoregulation during N starvation in the control of cell physiology and metabolism especially TAG accumulation. According to these findings, we propose that osmolarity should be carefully monitored during the industrial production of biodiesel.

Abstract Background Photosynthetic oleaginous microalgae are promising feedstocks for biofuels. Acyl-CoA:diacylglycerol acyltransferases (DGATs) represent rich sources for engineering microalgal lipid production. The principal activity of DGATs has been defined as a single-function enzyme catalyzing the esterification of diacylglycerol with acyl-CoA. Results A dual-function PtWS/DGAT associated with diatom Phaeodactylum tricornutum is discovered in the current study. Distinctive to documented microalgal DGAT types, PtWS/DGAT exhibits activities of both a wax ester synthase (WS) and a DGAT. WS/DGATs are broadly distributed in microalgae, with different topology and phylogeny from those of DGAT1s, DGAT2s, and DGAT3s. In vitro and in vivo assays revealed that PtWS/DGAT, functioning as either a WS or a DGAT, exhibited a preference on saturated FA substrate. Endogenous overexpression of PtWS/DGAT demonstrated that the DGAT activity was dominant, whereas the WS activity was condition dependent and relatively minor. Compared with the wild type (WT), overexpression of PtWS/DGAT in the diatom resulted in increased levels of total lipids (TL) and triacylglycerol (TAG) regardless of nitrogen availability. The stability and scalability of the introduced traits were further investigated at a 10-L photobioreactor, where the mutant growth resembled WT, with moderately increased productivity of TL and TAG. Furthermore, the production of wax esters increased considerably (from undetectable levels to 2.83%) under nitrogen-deplete conditions. Conclusions PtWS/DGAT is a bifunctional enzyme and may serve as a promising target for the engineering of microalga-based oils and waxes for future industrial use.

Background: Nitrogen (N) starvation in algae induces a variety of structural and metabolic changes including accumulation of triacylglycerol (TAG). Given the promising prospect of using algae as feedstock for biofuel production, accumulation of TAG upon N starvation becomes an ideal system to study TAG biosynthesis. Under nitrogen-depleted conditions, algae also accumulate compatible solutes such as sugar and certain amino acids, which is expected to elevate osmolarity in the cytoplasm. However, how osmoregulation is maintained and how it impacts on carbon metabolism, especially TAG accumulation under N starvation, are not well understood. Results: We show here that potassium channel KCN11 localized in the contractile vacuole (CV) mediates osmoregulation during N starvation and loss of KCN11 profoundly affects cell physiology and TAG biosynthesis. KCN11 level is increased and the CV pulsation is accelerated. Loss of KCN11 induces aberrant CV cycle, inhibition of cell growth, increase of cell size, inhibition of chlorophyll loss and TAG accumulation. These effects are rescued by addition of sucrose to raise osmolarity in the culture medium, indicating that osmoregulation is required for cell adaptation to N starvation. Metabolomic analysis shows reduction of acetyl-CoA and accumulation of glyceraldehyde-3-phosphate in kcn11 mutant relative to the control under N starvation, indicating that defects in acetyl-CoA biosynthesis and some metabolic steps from glyceraldehyde-3-phosphate to TAG contribute to the decreased TAG accumulation due to loss of osmoregulation. Conclusions: This work provides novel insight of osmoregulation during N starvation in the control of cell physiology and metabolism especially TAG accumulation. According to these findings, we propose that osmolarity should be carefully monitored during the industrial production of biodiesel. [ABSTRACT FROM AUTHOR]

Background: Photosynthetic oleaginous microalgae are promising feedstocks for biofuels. Acyl-CoA:diacylglycerol acyltransferases (DGATs) represent rich sources for engineering microalgal lipid production. The principal activity of DGATs has been defined as a single-function enzyme catalyzing the esterification of diacylglycerol with acyl-CoA., Results: A dual-function PtWS/DGAT associated with diatom Phaeodactylum tricornutum is discovered in the current study. Distinctive to documented microalgal DGAT types, PtWS/DGAT exhibits activities of both a wax ester synthase (WS) and a DGAT. WS/DGATs are broadly distributed in microalgae, with different topology and phylogeny from those of DGAT1s, DGAT2s, and DGAT3s. In vitro and in vivo assays revealed that PtWS/DGAT, functioning as either a WS or a DGAT, exhibited a preference on saturated FA substrate. Endogenous overexpression of PtWS/DGAT demonstrated that the DGAT activity was dominant, whereas the WS activity was condition dependent and relatively minor. Compared with the wild type (WT), overexpression of PtWS/DGAT in the diatom resulted in increased levels of total lipids (TL) and triacylglycerol (TAG) regardless of nitrogen availability. The stability and scalability of the introduced traits were further investigated at a 10-L photobioreactor, where the mutant growth resembled WT, with moderately increased productivity of TL and TAG. Furthermore, the production of wax esters increased considerably (from undetectable levels to 2.83%) under nitrogen-deplete conditions., Conclusions: PtWS/DGAT is a bifunctional enzyme and may serve as a promising target for the engineering of microalga-based oils and waxes for future industrial use.