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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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GC-MS analysis of the wax esters in E. gracilis Z under hypoxic-light conditions.(A) A total ion current (TIC) chromatogram from GC-MS analysis demonstrates wax ester peaks with the carbon chain lengths of C25, C26, C27, C28, C29, C30 and C31. (B) A mass spectral fragmentation pattern of the C28 wax ester displays parental ion (m/z 424.4) and fatty acid-specific fragment ions (m/z = 201.2, 215.2, 229.2, and 243.2 are attributable to C12:0, C13:0, C14:0 and C15:0 fatty acids, respectively).
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pone.0162827.g001: GC-MS analysis of the wax esters in E. gracilis Z under hypoxic-light conditions.(A) A total ion current (TIC) chromatogram from GC-MS analysis demonstrates wax ester peaks with the carbon chain lengths of C25, C26, C27, C28, C29, C30 and C31. (B) A mass spectral fragmentation pattern of the C28 wax ester displays parental ion (m/z 424.4) and fatty acid-specific fragment ions (m/z = 201.2, 215.2, 229.2, and 243.2 are attributable to C12:0, C13:0, C14:0 and C15:0 fatty acids, respectively).

Mentions: While E. gracilis Z contains only very small amounts of wax esters under aerobic conditions, GC-MS analysis of the nonpolar fraction from the hypoxic samples indicated that wax esters were abundantly synthesized under hypoxic-dark conditions [26]. Fig 1 shows the gas chromatogram and representative mass spectrum of wax esters accumulated in E. gracilis Z under hypoxic-light conditions. The detected wax esters were identified by comparing their retention times and mass spectral fragmentation patterns with those of the authentic standard compounds [26]. As shown in Fig 1A, the majority of wax esters contained between 26 and 30 carbon atoms. The mass spectrum of the most abundant component (Fig 1B, C28 wax ester) exhibited a main fragment at m/z 229, attributable to C14:0 fatty acid, together with a molecular ion peak at m/z 424; this dominant species was identified as a C28 wax ester composed of myristic acid and myristyl alcohol (C14:0-C14:0). Other wax ester components were identified in a similar manner based on their retention times and mass spectral fragmentation patterns, and C14:0-C14:0 was shown to be the dominant wax ester in all samples [26]. The total chain lengths of wax esters synthesized under hypoxic-light conditions ranged from 22 to 34 carbon atoms, and unsaturated wax ester species were not detected. The wax ester accumulation profile in this study was in agreement with those reported in the previous study on the streptomycin-bleached mutant of E. gracilis Z [6, 26].


Critical Involvement of Environmental Carbon Dioxide Fixation to Drive Wax Ester Fermentation in Euglena
GC-MS analysis of the wax esters in E. gracilis Z under hypoxic-light conditions.(A) A total ion current (TIC) chromatogram from GC-MS analysis demonstrates wax ester peaks with the carbon chain lengths of C25, C26, C27, C28, C29, C30 and C31. (B) A mass spectral fragmentation pattern of the C28 wax ester displays parental ion (m/z 424.4) and fatty acid-specific fragment ions (m/z = 201.2, 215.2, 229.2, and 243.2 are attributable to C12:0, C13:0, C14:0 and C15:0 fatty acids, respectively).
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pone.0162827.g001: GC-MS analysis of the wax esters in E. gracilis Z under hypoxic-light conditions.(A) A total ion current (TIC) chromatogram from GC-MS analysis demonstrates wax ester peaks with the carbon chain lengths of C25, C26, C27, C28, C29, C30 and C31. (B) A mass spectral fragmentation pattern of the C28 wax ester displays parental ion (m/z 424.4) and fatty acid-specific fragment ions (m/z = 201.2, 215.2, 229.2, and 243.2 are attributable to C12:0, C13:0, C14:0 and C15:0 fatty acids, respectively).
Mentions: While E. gracilis Z contains only very small amounts of wax esters under aerobic conditions, GC-MS analysis of the nonpolar fraction from the hypoxic samples indicated that wax esters were abundantly synthesized under hypoxic-dark conditions [26]. Fig 1 shows the gas chromatogram and representative mass spectrum of wax esters accumulated in E. gracilis Z under hypoxic-light conditions. The detected wax esters were identified by comparing their retention times and mass spectral fragmentation patterns with those of the authentic standard compounds [26]. As shown in Fig 1A, the majority of wax esters contained between 26 and 30 carbon atoms. The mass spectrum of the most abundant component (Fig 1B, C28 wax ester) exhibited a main fragment at m/z 229, attributable to C14:0 fatty acid, together with a molecular ion peak at m/z 424; this dominant species was identified as a C28 wax ester composed of myristic acid and myristyl alcohol (C14:0-C14:0). Other wax ester components were identified in a similar manner based on their retention times and mass spectral fragmentation patterns, and C14:0-C14:0 was shown to be the dominant wax ester in all samples [26]. The total chain lengths of wax esters synthesized under hypoxic-light conditions ranged from 22 to 34 carbon atoms, and unsaturated wax ester species were not detected. The wax ester accumulation profile in this study was in agreement with those reported in the previous study on the streptomycin-bleached mutant of E. gracilis Z [6, 26].

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