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

Adchara Padermshoke,Masanori Arita,Masatoshi Nakamoto,Shigehiko Kanaya,Atsushi Okazawa,Masami Nakazawa,Takumi Ogawa,Daisaku Ohta,Kazuki Nishio,Shihui Yang

doi:10.1371/journal.pone.0162827

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.

Highlights

Euglena gracilis is a unicellular flagellated protist that possesses chloroplasts, which allow its autotrophic growth, and it can grow as a heterotroph [1]
Paramylon and wax esters predominantly accumulate in normoxic-dark and hypoxic-dark cultures of E. gracilis Z, respectively, while proteins and other lipids are most abundant under normoxic-light conditions [27]
We studied the nature of the hypoxia-induced wax ester fermentation in E. gracilis Z under continuous illumination

Summary

Introduction

Euglena gracilis is a unicellular flagellated protist that possesses chloroplasts, which allow its autotrophic growth, and it can grow as a heterotroph [1]. It is common in freshwater and can withstand harsh conditions that are harmful to microorganisms [2, 3]. Transferring the aerobic culture to hypoxic conditions enables the synthesis of medium- to long-chain wax monoesters via a unique metabolic process called wax ester fermentation, along with the breakdown of the reserve paramylon. Switching the hypoxic culture back to aerobic conditions reverses the wax ester fermentation reactions [9]. The cellular adenosine triphosphate (ATP) content of Euglena decreases instantly upon exposure to hypoxic conditions, but gradually regains its original level when the wax ester fermentation becomes apparent and stays constant during the anaerobiosis; Euglena lives on respiration under normoxic conditions and on fermentation under hypoxic conditions [5]

Methods

Results

Discussion

Conclusion