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The small molecule fenpropimorph rapidly converts chloroplast membrane lipids to triacylglycerols in Chlamydomonas reinhardtii.

Kim H, Jang S, Kim S, Yamaoka Y, Hong D, Song WY, Nishida I, Li-Beisson Y, Lee Y - Front Microbiol (2015)

Bottom Line: Concern about global warming has prompted an intense interest in developing economical methods of producing biofuels.Microalgae provide a promising platform for biofuel production, because they accumulate high levels of lipids, and do not compete with food or feed sources.However, current methods of producing algal oil involve subjecting the microalgae to stress conditions, such as nitrogen deprivation, and are prohibitively expensive.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Life Sciences, Pohang University of Science and Technology Pohang, South Korea.

ABSTRACT
Concern about global warming has prompted an intense interest in developing economical methods of producing biofuels. Microalgae provide a promising platform for biofuel production, because they accumulate high levels of lipids, and do not compete with food or feed sources. However, current methods of producing algal oil involve subjecting the microalgae to stress conditions, such as nitrogen deprivation, and are prohibitively expensive. Here, we report that the fungicide fenpropimorph rapidly causes high levels of neutral lipids to accumulate in Chlamydomonas reinhardtii cells. When treated with fenpropimorph (10 μg mL(-1)) for 1 h, Chlamydomonas cells accumulated at least fourfold the amount of triacylglycerols (TAGs) present in the untreated control cells. Furthermore, the quantity of TAGs present after 1 h of fenpropimorph treatment was over twofold higher than that formed after 9 days of nitrogen starvation in medium with no acetate supplement. Biochemical analysis of lipids revealed that the accumulated TAGs were derived mainly from chloroplast polar membrane lipids. Such a conversion of chloroplast polar lipids to TAGs is desirable for biodiesel production, because polar lipids are usually removed during the biodiesel production process. Thus, our data exemplified that a cost and time effective method of producing TAGs is possible using fenpropimorph or similar drugs.

No MeSH data available.


Related in: MedlinePlus

Fenpropimorph induces neutral lipid accumulation in Chlamydomonas reinhardtii. (A) Fenpropimorph-induced LD formation occurs in a dose-dependent manner. The fluorescence intensity (FI) of a neutral lipid specific-dye, Nile red, was determined. Late mid-log phase Chlamydomonas cells (N+, acetate+) were treated with ethanol (solvent control) or fenpropimorph (1 h, at RT). Averages from three replicate experiments are presented. Bars represent SE. Significant differences, as determined by Student’s t-test, are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). (B) Fenpropimorph-induced TAGs were extracted and analyzed using biochemical methods. Control cells were treated with the same volume of ethanol used to dissolve fenpropimorph. Averages from triplicate experiments are presented. Bars represent SE. Significant differences, as determined by Student’s t-test, are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Images of Nile red-stained LD accumulation in fenpropimorph-treated cells. Cells were treated with fenpropimorph for 1 h. Images were obtained using a fluorescence microscope. (D) Time-dependent change in TAG concentration in fenpropimorph-treated Chlamydomonas cells. TAG accumulation induced by fenpropimorph (10 μg mL-1) treatment was analyzed biochemically. Averages and SE from three replicate experiments are presented. TAG levels shown were converted to μg from nmol values obtained from GC experiment. The original nmol values for each time point (5, 15, 45, 85, and 125 min) were 26.8 ± 4.7, 40.9 ± 0.4, 51.2 ± 5.1, 66.1 ± 24.2, and 27.7 ± 5.7, respectively, for control samples, and 65.4 ± 3.0, 120.7 ± 1.2, 255.0 ± 8.5, 260.7 ± 21.9, and 278.0 ± 1.0, respectively, for fenpropimorph-treated samples. In experiments shown in (A – D), Chlamydomonas cells in late mid-log phase culture in TAP medium (N+, acetate+) were used. (E,F) Comparison of the effect of nitrogen deprivation and fenpropimorph treatment on lipid induction efficiency in Chlamydomonas cells. (E) Nile red fluorescence intensity of control Chlamydomonas cells, and of cells subjected to fenpropimorph treatment (1 h, 25°C), and nitrogen deprivation (for the indicated number of days). Chlamydomonas cells were grown in normal conditions to mid-log phase, and washed to remove acetate and nitrogen from the medium. They were then re-suspended in TAP medium without an acetate or nitrogen source, and then either treated with ethanol (solvent control) or fenpropimorph (10 μg mL-1) for 1 h, or transferred to the nitrogen-deficient conditions and incubated for 3, 6, or 9 days. The FI value was measured. Averages from three replicate experiments are presented. Bars represent SE. (F) Biochemical analysis of TAG content in cells treated as in (E). Averages from three replicate experiments are presented. Bars represent SE.
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Figure 1: Fenpropimorph induces neutral lipid accumulation in Chlamydomonas reinhardtii. (A) Fenpropimorph-induced LD formation occurs in a dose-dependent manner. The fluorescence intensity (FI) of a neutral lipid specific-dye, Nile red, was determined. Late mid-log phase Chlamydomonas cells (N+, acetate+) were treated with ethanol (solvent control) or fenpropimorph (1 h, at RT). Averages from three replicate experiments are presented. Bars represent SE. Significant differences, as determined by Student’s t-test, are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). (B) Fenpropimorph-induced TAGs were extracted and analyzed using biochemical methods. Control cells were treated with the same volume of ethanol used to dissolve fenpropimorph. Averages from triplicate experiments are presented. Bars represent SE. Significant differences, as determined by Student’s t-test, are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Images of Nile red-stained LD accumulation in fenpropimorph-treated cells. Cells were treated with fenpropimorph for 1 h. Images were obtained using a fluorescence microscope. (D) Time-dependent change in TAG concentration in fenpropimorph-treated Chlamydomonas cells. TAG accumulation induced by fenpropimorph (10 μg mL-1) treatment was analyzed biochemically. Averages and SE from three replicate experiments are presented. TAG levels shown were converted to μg from nmol values obtained from GC experiment. The original nmol values for each time point (5, 15, 45, 85, and 125 min) were 26.8 ± 4.7, 40.9 ± 0.4, 51.2 ± 5.1, 66.1 ± 24.2, and 27.7 ± 5.7, respectively, for control samples, and 65.4 ± 3.0, 120.7 ± 1.2, 255.0 ± 8.5, 260.7 ± 21.9, and 278.0 ± 1.0, respectively, for fenpropimorph-treated samples. In experiments shown in (A – D), Chlamydomonas cells in late mid-log phase culture in TAP medium (N+, acetate+) were used. (E,F) Comparison of the effect of nitrogen deprivation and fenpropimorph treatment on lipid induction efficiency in Chlamydomonas cells. (E) Nile red fluorescence intensity of control Chlamydomonas cells, and of cells subjected to fenpropimorph treatment (1 h, 25°C), and nitrogen deprivation (for the indicated number of days). Chlamydomonas cells were grown in normal conditions to mid-log phase, and washed to remove acetate and nitrogen from the medium. They were then re-suspended in TAP medium without an acetate or nitrogen source, and then either treated with ethanol (solvent control) or fenpropimorph (10 μg mL-1) for 1 h, or transferred to the nitrogen-deficient conditions and incubated for 3, 6, or 9 days. The FI value was measured. Averages from three replicate experiments are presented. Bars represent SE. (F) Biochemical analysis of TAG content in cells treated as in (E). Averages from three replicate experiments are presented. Bars represent SE.

Mentions: Chlamydomonas cells were treated with 10 μg mL-1 of fenpropimorph, and the cellular oil content was tracked by monitoring the fluorescence intensity (FI) of Nile red, a lipophilic dye specific for neutral lipids (Kou et al., 2013). After just 1 h of fenpropimorph treatment, the FI increased dramatically (Figure 1A). Furthermore, the FI increased as the concentrations of fenpropimorph increased from 5 to 20 μg mL-1 (Figure 1A). Biochemical analyses of lipids extracted from fenpropimorph-treated cells revealed that TAGs increased 6–15-fold in fenpropimorph-treated versus control Chlamydomonas cells, in proportion to the increases in drug concentrations from 5 to 20 μg mL-1 (Figure 1B). The fold increase values varied between experiments, perhaps due to slight differences in culture age (within the late mid-log phase), and consequent variation in the TAG levels of the control samples which ranged between 40 and 135 nmol fatty acids in TAG per 6 × 107cells. The effect of the drug was maximal at the late mid-log phase (at 4 days of culture under our conditions), and slightly less at the stationary phase (Figure A1). Microscopic observations revealed that fenpropimorph-treated cells had more LDs than did the control cells (Figure 1C). The effect of fenpropimorph treatment on TAG induction in Chlamydomonas was very rapid: TAG levels increased significantly after as little as 5 min of treatment with 10 μg mL-1 fenpropimorph, and became saturated at 45–85 min (i.e., longer treatment did not induce further TAG accumulation; Figure 1D).


The small molecule fenpropimorph rapidly converts chloroplast membrane lipids to triacylglycerols in Chlamydomonas reinhardtii.

Kim H, Jang S, Kim S, Yamaoka Y, Hong D, Song WY, Nishida I, Li-Beisson Y, Lee Y - Front Microbiol (2015)

Fenpropimorph induces neutral lipid accumulation in Chlamydomonas reinhardtii. (A) Fenpropimorph-induced LD formation occurs in a dose-dependent manner. The fluorescence intensity (FI) of a neutral lipid specific-dye, Nile red, was determined. Late mid-log phase Chlamydomonas cells (N+, acetate+) were treated with ethanol (solvent control) or fenpropimorph (1 h, at RT). Averages from three replicate experiments are presented. Bars represent SE. Significant differences, as determined by Student’s t-test, are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). (B) Fenpropimorph-induced TAGs were extracted and analyzed using biochemical methods. Control cells were treated with the same volume of ethanol used to dissolve fenpropimorph. Averages from triplicate experiments are presented. Bars represent SE. Significant differences, as determined by Student’s t-test, are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Images of Nile red-stained LD accumulation in fenpropimorph-treated cells. Cells were treated with fenpropimorph for 1 h. Images were obtained using a fluorescence microscope. (D) Time-dependent change in TAG concentration in fenpropimorph-treated Chlamydomonas cells. TAG accumulation induced by fenpropimorph (10 μg mL-1) treatment was analyzed biochemically. Averages and SE from three replicate experiments are presented. TAG levels shown were converted to μg from nmol values obtained from GC experiment. The original nmol values for each time point (5, 15, 45, 85, and 125 min) were 26.8 ± 4.7, 40.9 ± 0.4, 51.2 ± 5.1, 66.1 ± 24.2, and 27.7 ± 5.7, respectively, for control samples, and 65.4 ± 3.0, 120.7 ± 1.2, 255.0 ± 8.5, 260.7 ± 21.9, and 278.0 ± 1.0, respectively, for fenpropimorph-treated samples. In experiments shown in (A – D), Chlamydomonas cells in late mid-log phase culture in TAP medium (N+, acetate+) were used. (E,F) Comparison of the effect of nitrogen deprivation and fenpropimorph treatment on lipid induction efficiency in Chlamydomonas cells. (E) Nile red fluorescence intensity of control Chlamydomonas cells, and of cells subjected to fenpropimorph treatment (1 h, 25°C), and nitrogen deprivation (for the indicated number of days). Chlamydomonas cells were grown in normal conditions to mid-log phase, and washed to remove acetate and nitrogen from the medium. They were then re-suspended in TAP medium without an acetate or nitrogen source, and then either treated with ethanol (solvent control) or fenpropimorph (10 μg mL-1) for 1 h, or transferred to the nitrogen-deficient conditions and incubated for 3, 6, or 9 days. The FI value was measured. Averages from three replicate experiments are presented. Bars represent SE. (F) Biochemical analysis of TAG content in cells treated as in (E). Averages from three replicate experiments are presented. Bars represent SE.
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Figure 1: Fenpropimorph induces neutral lipid accumulation in Chlamydomonas reinhardtii. (A) Fenpropimorph-induced LD formation occurs in a dose-dependent manner. The fluorescence intensity (FI) of a neutral lipid specific-dye, Nile red, was determined. Late mid-log phase Chlamydomonas cells (N+, acetate+) were treated with ethanol (solvent control) or fenpropimorph (1 h, at RT). Averages from three replicate experiments are presented. Bars represent SE. Significant differences, as determined by Student’s t-test, are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). (B) Fenpropimorph-induced TAGs were extracted and analyzed using biochemical methods. Control cells were treated with the same volume of ethanol used to dissolve fenpropimorph. Averages from triplicate experiments are presented. Bars represent SE. Significant differences, as determined by Student’s t-test, are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Images of Nile red-stained LD accumulation in fenpropimorph-treated cells. Cells were treated with fenpropimorph for 1 h. Images were obtained using a fluorescence microscope. (D) Time-dependent change in TAG concentration in fenpropimorph-treated Chlamydomonas cells. TAG accumulation induced by fenpropimorph (10 μg mL-1) treatment was analyzed biochemically. Averages and SE from three replicate experiments are presented. TAG levels shown were converted to μg from nmol values obtained from GC experiment. The original nmol values for each time point (5, 15, 45, 85, and 125 min) were 26.8 ± 4.7, 40.9 ± 0.4, 51.2 ± 5.1, 66.1 ± 24.2, and 27.7 ± 5.7, respectively, for control samples, and 65.4 ± 3.0, 120.7 ± 1.2, 255.0 ± 8.5, 260.7 ± 21.9, and 278.0 ± 1.0, respectively, for fenpropimorph-treated samples. In experiments shown in (A – D), Chlamydomonas cells in late mid-log phase culture in TAP medium (N+, acetate+) were used. (E,F) Comparison of the effect of nitrogen deprivation and fenpropimorph treatment on lipid induction efficiency in Chlamydomonas cells. (E) Nile red fluorescence intensity of control Chlamydomonas cells, and of cells subjected to fenpropimorph treatment (1 h, 25°C), and nitrogen deprivation (for the indicated number of days). Chlamydomonas cells were grown in normal conditions to mid-log phase, and washed to remove acetate and nitrogen from the medium. They were then re-suspended in TAP medium without an acetate or nitrogen source, and then either treated with ethanol (solvent control) or fenpropimorph (10 μg mL-1) for 1 h, or transferred to the nitrogen-deficient conditions and incubated for 3, 6, or 9 days. The FI value was measured. Averages from three replicate experiments are presented. Bars represent SE. (F) Biochemical analysis of TAG content in cells treated as in (E). Averages from three replicate experiments are presented. Bars represent SE.
Mentions: Chlamydomonas cells were treated with 10 μg mL-1 of fenpropimorph, and the cellular oil content was tracked by monitoring the fluorescence intensity (FI) of Nile red, a lipophilic dye specific for neutral lipids (Kou et al., 2013). After just 1 h of fenpropimorph treatment, the FI increased dramatically (Figure 1A). Furthermore, the FI increased as the concentrations of fenpropimorph increased from 5 to 20 μg mL-1 (Figure 1A). Biochemical analyses of lipids extracted from fenpropimorph-treated cells revealed that TAGs increased 6–15-fold in fenpropimorph-treated versus control Chlamydomonas cells, in proportion to the increases in drug concentrations from 5 to 20 μg mL-1 (Figure 1B). The fold increase values varied between experiments, perhaps due to slight differences in culture age (within the late mid-log phase), and consequent variation in the TAG levels of the control samples which ranged between 40 and 135 nmol fatty acids in TAG per 6 × 107cells. The effect of the drug was maximal at the late mid-log phase (at 4 days of culture under our conditions), and slightly less at the stationary phase (Figure A1). Microscopic observations revealed that fenpropimorph-treated cells had more LDs than did the control cells (Figure 1C). The effect of fenpropimorph treatment on TAG induction in Chlamydomonas was very rapid: TAG levels increased significantly after as little as 5 min of treatment with 10 μg mL-1 fenpropimorph, and became saturated at 45–85 min (i.e., longer treatment did not induce further TAG accumulation; Figure 1D).

Bottom Line: Concern about global warming has prompted an intense interest in developing economical methods of producing biofuels.Microalgae provide a promising platform for biofuel production, because they accumulate high levels of lipids, and do not compete with food or feed sources.However, current methods of producing algal oil involve subjecting the microalgae to stress conditions, such as nitrogen deprivation, and are prohibitively expensive.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Life Sciences, Pohang University of Science and Technology Pohang, South Korea.

ABSTRACT
Concern about global warming has prompted an intense interest in developing economical methods of producing biofuels. Microalgae provide a promising platform for biofuel production, because they accumulate high levels of lipids, and do not compete with food or feed sources. However, current methods of producing algal oil involve subjecting the microalgae to stress conditions, such as nitrogen deprivation, and are prohibitively expensive. Here, we report that the fungicide fenpropimorph rapidly causes high levels of neutral lipids to accumulate in Chlamydomonas reinhardtii cells. When treated with fenpropimorph (10 μg mL(-1)) for 1 h, Chlamydomonas cells accumulated at least fourfold the amount of triacylglycerols (TAGs) present in the untreated control cells. Furthermore, the quantity of TAGs present after 1 h of fenpropimorph treatment was over twofold higher than that formed after 9 days of nitrogen starvation in medium with no acetate supplement. Biochemical analysis of lipids revealed that the accumulated TAGs were derived mainly from chloroplast polar membrane lipids. Such a conversion of chloroplast polar lipids to TAGs is desirable for biodiesel production, because polar lipids are usually removed during the biodiesel production process. Thus, our data exemplified that a cost and time effective method of producing TAGs is possible using fenpropimorph or similar drugs.

No MeSH data available.


Related in: MedlinePlus