Fig 1.
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).
Fig 2.
Major wax ester components in hypoxic E. gracilis Z cells.
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.
Fig 3.
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.
Table 1.
Accumulation profiles of the five major wax ester species in E. gracilis Z under hypoxic, anoxic + CO2, and anoxic + CO2 + NaHCO3 conditions.
Fig 4.
Recovery of the inhibited wax ester fermentation in anoxia by inorganic carbon sources.
The relative levels of C28 (14:0–14:0) are shown as representing the wax esters in E. gracilis 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.
Table 2.
Incorporation of externally-added 13C into wax esters in E. gracilis Z.
Fig 5.
13C-labeling time course of organic acids in the presence or absence of 3MPA.
(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 t-test with Bonferroni correction; statistical significance between treatments at each time point (green); Student’s t-test; † p < 0.10, *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 6.
Wax ester fermentation in E. gracilis.
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 [8, 15, 29, 31]. 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. Abbreviations for key enzymes: 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 Euglena to produce isocitrate from α-KG.