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Critical Involvement of Environmental Carbon Dioxide Fixation to Drive Wax Ester Fermentation in Euglena

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ABSTRACT

Accumulation profiles of wax esters in Euglena gracilis Z were studied under several environmental conditions. The highest amount of total wax esters accumulated under hypoxia in the dark, and C28 (myristyl-myristate, C14:0-C14:0) was prevalent among all conditions investigated. The wax ester production was almost completely suppressed under anoxia in the light, and supplying exogenous inorganic carbon sources restored wax ester fermentation, indicating the need for external carbon sources for the wax ester fermentation. 13C-labeling experiments revealed specific isotopic enrichment in the odd-numbered fatty acids derived from wax esters, indicating that the exogenously-supplied CO2 was incorporated into wax esters via the propionyl-CoA pathway through the reverse tricarboxylic acid (TCA) cycle. The addition of 3-mercaptopicolinic acid, a phosphoenolpyruvate carboxykinase (PEPCK) inhibitor, significantly affected the incorporation of 13C into citrate and malate as the biosynthetic intermediates of the odd-numbered fatty acids, suggesting the involvement of PEPCK reaction to drive wax ester fermentation. Additionally, the 13C-enrichment pattern of succinate suggested that the CO2 assimilation might proceed through alternative pathways in addition to the PEPCK reaction. The current results indicate that the mechanisms of anoxic CO2 assimilation are an important target to reinforce wax ester fermentation in Euglena.

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Inhibition of the wax ester fermentation in anoxia.The wax ester accumulation was almost completely abolished under anoxic-light conditions, as shown by the levels of C28 (14:0–14:0), which represents wax esters. The light-grown cells were subjected to anoxic and hypoxic conditions under both light and dark conditions. Hypoxia and anoxia were induced by stopping the culture agitation and N2 aeration (10 min), respectively. The results under light-anoxic conditions are magnified in the inset. Error bars indicate standard deviation from triplicate cultures.
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pone.0162827.g003: Inhibition of the wax ester fermentation in anoxia.The wax ester accumulation was almost completely abolished under anoxic-light conditions, as shown by the levels of C28 (14:0–14:0), which represents wax esters. The light-grown cells were subjected to anoxic and hypoxic conditions under both light and dark conditions. Hypoxia and anoxia were induced by stopping the culture agitation and N2 aeration (10 min), respectively. The results under light-anoxic conditions are magnified in the inset. Error bars indicate standard deviation from triplicate cultures.

Mentions: We have, so far, studied wax ester fermentation in E. gracilis Z under hypoxic conditions initiated by stopping the shaking culture (Figs 1 and 2). To evaluate the metabolism under strictly anaerobic conditions, anoxic cells were prepared by bubbling N2 gas into the culture medium for 10 min. Then the cells were incubated either in the light or in the dark for additional 24 h before harvested. The pH of the culture medium before and after N2 aeration was 7.0 ± 0.5 and 7.7 ± 0.2, respectively. Despite fluctuations among experimental replicates after the 24-h incubation, the medium pH consistently increased to 9.0 ± 0.5 in the light, but stayed within the range of 7.0 ± 0.5 after dark incubation. Fig 3 shows the effects of light conditions on the accumulation levels of the dominant C28 wax ester, representing the general wax ester profile in E. gracilis Z, under both anoxic (N2 gas aeration) and hypoxic conditions (stopped shaking the culture). Irrespective of hypoxia and anoxia, the amount of wax ester accumulation was considerably higher in the dark-grown cells than the light-grown cells. We also observed a strikingly different result under anoxic-light conditions: the wax ester fermentation was nearly completely suppressed, while it was apparently unaffected in dark conditions (Fig 3). As described above, the culture medium pH increased during anoxic-light cultivation, where wax ester accumulation was greatly suppressed. The wax ester accumulation levels did not recover during 24-h of the anoxic-light, even when the initial pH 7.0 of the culture medium fluctuated within the range of pH 7.0 ± 0.5 in the presence of either HEPES or BES, suggesting that the inhibited wax ester accumulation was not simply a result of the increase in the medium pH.


Critical Involvement of Environmental Carbon Dioxide Fixation to Drive Wax Ester Fermentation in Euglena
Inhibition of the wax ester fermentation in anoxia.The wax ester accumulation was almost completely abolished under anoxic-light conditions, as shown by the levels of C28 (14:0–14:0), which represents wax esters. The light-grown cells were subjected to anoxic and hypoxic conditions under both light and dark conditions. Hypoxia and anoxia were induced by stopping the culture agitation and N2 aeration (10 min), respectively. The results under light-anoxic conditions are magnified in the inset. Error bars indicate standard deviation from triplicate cultures.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036851&req=5

pone.0162827.g003: Inhibition of the wax ester fermentation in anoxia.The wax ester accumulation was almost completely abolished under anoxic-light conditions, as shown by the levels of C28 (14:0–14:0), which represents wax esters. The light-grown cells were subjected to anoxic and hypoxic conditions under both light and dark conditions. Hypoxia and anoxia were induced by stopping the culture agitation and N2 aeration (10 min), respectively. The results under light-anoxic conditions are magnified in the inset. Error bars indicate standard deviation from triplicate cultures.
Mentions: We have, so far, studied wax ester fermentation in E. gracilis Z under hypoxic conditions initiated by stopping the shaking culture (Figs 1 and 2). To evaluate the metabolism under strictly anaerobic conditions, anoxic cells were prepared by bubbling N2 gas into the culture medium for 10 min. Then the cells were incubated either in the light or in the dark for additional 24 h before harvested. The pH of the culture medium before and after N2 aeration was 7.0 ± 0.5 and 7.7 ± 0.2, respectively. Despite fluctuations among experimental replicates after the 24-h incubation, the medium pH consistently increased to 9.0 ± 0.5 in the light, but stayed within the range of 7.0 ± 0.5 after dark incubation. Fig 3 shows the effects of light conditions on the accumulation levels of the dominant C28 wax ester, representing the general wax ester profile in E. gracilis Z, under both anoxic (N2 gas aeration) and hypoxic conditions (stopped shaking the culture). Irrespective of hypoxia and anoxia, the amount of wax ester accumulation was considerably higher in the dark-grown cells than the light-grown cells. We also observed a strikingly different result under anoxic-light conditions: the wax ester fermentation was nearly completely suppressed, while it was apparently unaffected in dark conditions (Fig 3). As described above, the culture medium pH increased during anoxic-light cultivation, where wax ester accumulation was greatly suppressed. The wax ester accumulation levels did not recover during 24-h of the anoxic-light, even when the initial pH 7.0 of the culture medium fluctuated within the range of pH 7.0 ± 0.5 in the presence of either HEPES or BES, suggesting that the inhibited wax ester accumulation was not simply a result of the increase in the medium pH.

View Article: PubMed Central - PubMed

ABSTRACT

Accumulation profiles of wax esters in Euglena gracilis Z were studied under several environmental conditions. The highest amount of total wax esters accumulated under hypoxia in the dark, and C28 (myristyl-myristate, C14:0-C14:0) was prevalent among all conditions investigated. The wax ester production was almost completely suppressed under anoxia in the light, and supplying exogenous inorganic carbon sources restored wax ester fermentation, indicating the need for external carbon sources for the wax ester fermentation. 13C-labeling experiments revealed specific isotopic enrichment in the odd-numbered fatty acids derived from wax esters, indicating that the exogenously-supplied CO2 was incorporated into wax esters via the propionyl-CoA pathway through the reverse tricarboxylic acid (TCA) cycle. The addition of 3-mercaptopicolinic acid, a phosphoenolpyruvate carboxykinase (PEPCK) inhibitor, significantly affected the incorporation of 13C into citrate and malate as the biosynthetic intermediates of the odd-numbered fatty acids, suggesting the involvement of PEPCK reaction to drive wax ester fermentation. Additionally, the 13C-enrichment pattern of succinate suggested that the CO2 assimilation might proceed through alternative pathways in addition to the PEPCK reaction. The current results indicate that the mechanisms of anoxic CO2 assimilation are an important target to reinforce wax ester fermentation in Euglena.

No MeSH data available.


Related in: MedlinePlus