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

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

Accumulation profiles of the five major wax ester species in <i>E</i>. <i>gracilis</i> Z under hypoxic, anoxic + CO₂, and anoxic + CO₂ + NaHCO₃ conditions.

<p>Accumulation profiles of the five major wax ester species in <i>E</i>. <i>gracilis</i> Z under hypoxic, anoxic + CO₂, and anoxic + CO₂ + NaHCO₃ conditions.</p

¹³C-labeling time course of organic acids in the presence or absence of 3MPA.

<p>(A) Relative peak areas of organic acids in the presence (black symbol) or absence (white symbol) of 3MPA. (B) Relative abundances of M, M+1 and M+2 ions characteristic to each organic acid in the presence (3MPA-treated cells, black symbol) or absence (control cells, white symbol) of 3MPA. Values and error bars are the mean and standard deviation from triplicate cultures. Asterisks and daggers indicate statistically significant differences. Red (3MPA treatment) and blue (control) indicate statistically significant differences between 0 min and respective time points for each treatment. Student’s <i>t</i>-test with Bonferroni correction; statistical significance between treatments at each time point (green); Student’s <i>t</i>-test; † <i>p</i> < 0.10, *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p

Major wax ester components in hypoxic <i>E</i>. <i>gracilis</i> Z cells.

<p>Light-grown cells in the late log phase were transferred to hypoxic culture by stopping the culture agitation. Error bars indicate standard deviation from triplicate cultures.</p

Wax ester fermentation in <i>E</i>. <i>gracilis</i>.

<p>Under anaerobic conditions, mitochondrial wax ester fermentation proceeds through two routes: the C2-donor route supplies acetyl-CoA via the pyruvate:NADP⁺ oxidoreductase (PNO) reaction, and the C3-donor route includes anaerobic fumarate respiration to produce propionyl-CoA. The ¹³C-isotope from ¹³CO₂ (red circle) is incorporated into phosphoenolpyruvate (PEP) by the PEPCK reaction, and it is retained in propionyl-CoA that is used as the C3-donor in wax ester fermentation. The metabolic flow functioning under anaerobic conditions is indicated by green arrows [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162827#pone.0162827.ref008" target="_blank">8</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162827#pone.0162827.ref015" target="_blank">15</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162827#pone.0162827.ref029" target="_blank">29</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162827#pone.0162827.ref031" target="_blank">31</a>]. Another possible route for the carboxylation reaction is indicated by broken green arrows: α-ketoglutarate (α-KG) is carboxylated to yield citrate which ultimately serves as the precursor to produce oxaloacetate (OAA) in cytoplasm. <b>Abbreviations for key enzymes:</b> ACL, ATP citrate lyase; FUM, fumarase; FRD, fumarate reductase; IDH*, isocitrate dehydrogenase; KAT, ketoacyl-CoA thiolase; α-KGDH, α-ketoglutarate decarboxylase; MDH, malate dehydrogenase; ME, malic enzyme; MMC, methylmalonyl-CoA mutase; PK, pyruvate kinase; PNO, pyruvate:NADP⁺ oxidoreductase; PrCC, propionyl-CoA carboxylase; SSDH, succynyl semialdehyde dehydrogenase; RQ, rhodoquinone; SCS, succinyl-CoA synthetase. Abbreviations for metabolic intermediates: Cit, citrate; Fum, fumarate; Isocit, isocitorate; Mal, malate; OAA, oxaloacetate; Pyr, pyruvate; Suc, succinate; SucSA, succinate semialdehyde. *IDH has not been known in <i>Euglena</i> to produce isocitrate from α-KG.</p

Incorporation of externally-added ¹³C into wax esters in <i>E</i>. <i>gracilis</i> Z.

<p>Incorporation of externally-added ¹³C into wax esters in <i>E</i>. <i>gracilis</i> Z.</p

GC-MS analysis of the wax esters in <i>E</i>. <i>gracilis</i> Z under hypoxic-light conditions.

<p>(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 (<i>m</i>/<i>z</i> 424.4) and fatty acid-specific fragment ions (<i>m</i>/<i>z</i> = 201.2, 215.2, 229.2, and 243.2 are attributable to C12:0, C13:0, C14:0 and C15:0 fatty acids, respectively).</p

Recovery of the inhibited wax ester fermentation in anoxia by inorganic carbon sources.

<p>The relative levels of C28 (14:0–14:0) are shown as representing the wax esters in <i>E</i>. <i>gracilis</i> Z. The cells were cultivated in the light under hypoxic and anoxic conditions in the presence of CO₂ and/or NaHCO₃ (either 10 mM or 20 mM). The inset indicates light-anoxic conditions without an exogenous carbon supply. Error bars indicate standard deviation from triplicate cultures.</p