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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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0162827.g006: 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.

Mentions: To clarify the essential role of inorganic carbon source assimilation, we performed 13CO2-feeding experiments and demonstrated that the 13C-stable isotope was primarily incorporated into wax ester odd-numbered fatty acid moieties. This is consistent with the findings from 14C-radioisotope-labeling experiments. Schneider and Betz [23] reported that H14CO3- as well as [1,4-14C] succinate and [3-14C] propionate were preferentially incorporated into odd-numbered fatty acids and alcohols in anaerobically-grown E. gracilis T under dark conditions. In addition, it has been reported that [2-14C] pyruvate was not anaerobically incorporated into succinate, indicating that pyruvate carboxylase might not be primarily involved in the CO2 fixation into wax esters via OAA production [23]. Instead, the considerable decrease in 2-phosphoglycerate, pyruvate and PEP levels in fermenting E. gracilis Z under anaerobic-dark conditions [27] suggests the involvement of PEP in wax ester biosynthesis. Thus, upon incorporation of CO2 as shown in Fig 6, PEP is converted to OAA, which subsequently undergoes the reverse TCA cycle to yield succinyl-CoA and finally propionyl-CoA via the methylmalonyl-CoA intermediate [23]. Additionally, in the reverse TCA cycle, α-KG could be carboxylated to give isocitrate, which serves as a TCA cycle intermediate (Fig 6). However, α-KG carboxylation involved in wax ester fermentation has not been demonstrated in Euglena.


Critical Involvement of Environmental Carbon Dioxide Fixation to Drive Wax Ester Fermentation in Euglena
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.
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pone.0162827.g006: 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.
Mentions: To clarify the essential role of inorganic carbon source assimilation, we performed 13CO2-feeding experiments and demonstrated that the 13C-stable isotope was primarily incorporated into wax ester odd-numbered fatty acid moieties. This is consistent with the findings from 14C-radioisotope-labeling experiments. Schneider and Betz [23] reported that H14CO3- as well as [1,4-14C] succinate and [3-14C] propionate were preferentially incorporated into odd-numbered fatty acids and alcohols in anaerobically-grown E. gracilis T under dark conditions. In addition, it has been reported that [2-14C] pyruvate was not anaerobically incorporated into succinate, indicating that pyruvate carboxylase might not be primarily involved in the CO2 fixation into wax esters via OAA production [23]. Instead, the considerable decrease in 2-phosphoglycerate, pyruvate and PEP levels in fermenting E. gracilis Z under anaerobic-dark conditions [27] suggests the involvement of PEP in wax ester biosynthesis. Thus, upon incorporation of CO2 as shown in Fig 6, PEP is converted to OAA, which subsequently undergoes the reverse TCA cycle to yield succinyl-CoA and finally propionyl-CoA via the methylmalonyl-CoA intermediate [23]. Additionally, in the reverse TCA cycle, α-KG could be carboxylated to give isocitrate, which serves as a TCA cycle intermediate (Fig 6). However, α-KG carboxylation involved in wax ester fermentation has not been demonstrated in Euglena.

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