Under anaerobic conditions, Euglena gracilis produces a large amount of wax ester through mitochondrial fatty acid synthesis from storage polysaccharides termed paramylon, to generate ATP. Trans-2-enoyl-CoA reductases (TERs) in mitochondria have been considered to play a key role in this process, because the enzymes catalyze the reduction of short chain length CoA-substrates (such as crotonyl-CoA). A TER enzyme (EgTER1) has been previously identified and enzymologically characterized; however, its physiological significance remained to be evaluated by genetic analysis. We herein generated EgTER1-knockdown Euglena cells, in which total crotonyl-CoA reductase activity was decreased to 10% of control value. Notably, the knockdown cells showed a severe bleaching phenotype with deficiencies in chlorophylls and glycolipids, but grew normally under heterotrophic conditions (with glucose supplementation). Moreover, the knockdown cells accumulated much greater quantities of wax ester than control cells before and after transfer to anaerobic conditions, which was accompanied by a large metabolomic change. Furthermore, we failed to find any contribution of other potential TER genes in wax ester production. Our findings propose a novel role of EgTER1 in the greening process and demonstrate that this enzyme is dispensable for wax ester production under anaerobic conditions.
Citation: Tomiyama T, Goto K, Tanaka Y, Maruta T, Ogawa T, Sawa Y, et al. (2019) A major isoform of mitochondrial trans-2-enoyl-CoA reductase is dispensable for wax ester production in Euglena gracilis under anaerobic conditions. PLoS ONE 14(1): e0210755. https://doi.org/10.1371/journal.pone.0210755
Editor: Juan J. Loor, University of Illinois, UNITED STATES
Received: September 6, 2018; Accepted: December 30, 2018; Published: January 16, 2019
Copyright: © 2019 Tomiyama et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This study was supported by the Core Research for Evolutional Science and Technology (CREST) Program of the Japan Science and Technology Agency (JST) (YT, TM, TO, TI), http://www.jst.go.jp/presto/bioenergy/english/index.html. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Euglena gracilis is a single-celled eukaryotic alga containing chloroplasts with photosynthetic activity. Photosynthetic products are stored as polysaccharides termed paramylon, which consist of only liner β-1,3-glucan, in Euglena grown under aerobic conditions [1,2]. Notably, paramylon content often exceeds 50% of the dry weight of the cell [3,4]. Recently, one of two glucan synthase-like proteins, EgGSL2, has been found to be required for paramylon synthesis . When Euglena cells are placed in anaerobiosis, paramylon is rapidly degraded and used for the synthesis of wax esters. Wax esters produced by Euglena consist of saturated fatty acids and alcohols with carbon chain-lengths of 10 through 18 including odd-numbered chain lengths, with the major constituents being myristic acid (C14:0) and myristyl alcohol (C14:0) . The process of converting paramylon to wax esters involves glycolysis that produces ATP from substrate-level phosphorylation; therefore, this phenomenon has been termed “wax ester fermentation” [7,8].
At least three pathways for fatty acid synthesis have been proposed in Euglena: 1) via a multifunctional fatty acid synthase (FAS I) in the cytosol, 2) an acyl carrier protein-dependent system (FAS II) in illuminated chloroplasts, and 3) malonyl-CoA-independent fatty acid synthesis in mitochondria. The cytosolic and chloroplastic pathways require malonyl-CoA, which is synthesized in an ATP-dependent manner [9–11]. In contrast, the mitochondrial pathway does not require malonyl-CoA and can synthesize fatty acids with acetyl-CoA as both primer and C2 donor, using NAD(P)H as an electron donor . Thus, the mitochondrial system operates in an ATP-independent manner, allowing the net gain of ATP during the wax ester fermentation process.
Generally, the fatty acid synthetic pathway in mitochondria is the reverse of the ß-oxidation pathway, with one exception being that trans-2-enoyl-CoA reductase (TER) is used instead of acyl-CoA dehydrogenase, which is considered to catalyze irreversible reaction in the β-oxidation pathway (Fig 1). Instead, the Euglena TER can preferably catalyze the reduction of short chain length substrates (such as crotonyl-CoA). This feature allows the mitochondrial system to act as a de novo fatty acid synthetic pathway. Among the TER isoforms, one was previously purified from Euglena mitochondria and its cDNA sequence was determined . This enzyme, herein termed EgTER1 (see below), exhibited a preferential activity toward crotonyl-CoA using both NADH and NADPH as electron donors . TER activity in Euglena was found to be extremely low compared to that of the other enzymes in the mitochondrial fatty acid synthesis pathway (i.e., 3-ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase and trans-2-enoyl-CoA hydratase) [12–14]. In addition, TER activity was expected to be important for NAD+ supply for the glycolytic pathway. For these reasons, TER was considered as the rate-limiting step in wax ester fermentation. However, this scenario has been proposed only based on biochemical studies without any molecular cell biological evidence.
Following glycolysis in the cytosol, fatty acids are synthesized by the reverse reaction of β-oxidation as highlighted in the right side of the figure. trans-Enoyl-CoA reductase plays a key role in the fatty acid synthesis, rather than acyl-CoA dehydrogenase.
Accordingly, the aim of this study was to investigate the physiological significance of TER enzyme(s) in wax ester fermentation using a reverse genetic approach. Herein we succeeded in generating Euglena cells exhibiting knock down of the EgTER1 gene but, unexpectedly, failed to find any contribution of the enzyme to wax ester production under anaerobic conditions. Rather, we found this enzyme to be involved in greening process (chlorophyll synthesis and/or chloroplast development).
Materials and methods
Strain and culture
E. gracilis strain Z was grown in Koren-Hutner (KH) medium for heterotrophic growth  or Cramer-Myers (CM) medium for autotrophic growth  under continuous light conditions (50 μmol m-2 s-1) at 26°C for 6–14 days, by which time the stationary phase was reached. E. gracilis SM-ZK, a non-photosynthetic mutant strain , was cultured in KH medium with aeration at 26 ˚C for 6 days to the late logarithmic phase of growth. Aerobic cultures were grown with continuous shaking (120 rpm) under atmospheric conditions, and the anaerobic cultures were completely sealed and allowed to stand for 24 h after the replacement of air by N2 gas. Cell number and volume were determined using an electric field multi-channel cell counting system, CASY (Roche Diagnostics, Basel, Switzerland).
Gene knockdown experiments
Knockdown of TER genes was performed as described previously [5,18]. Template cDNAs were polymerase chain reaction (PCR)-amplified using primers including the T7 RNA polymerase promoter sequence at one end as listed in S1 Table. Then, the dsRNAs were synthesized from the template cDNAs using the MEGAscript RNAi Kit (Thermo Fisher Scientific, MA) by following the manufacturer’s instructions. Euglena cells of 2-d-old cultures were collected and resuspended in culture medium containing 4.2 mM Ca(NO3)2, 3.7 mM KH2PO4, and 2.1 mM MgSO4. The cell suspension (100 μL; approximately 5 × 106 cells) was transferred into a 0.4-cm-gap cuvette and electroporated with 5 μL of RNA solution (15 μg of dsRNA in 50 mM Tris-HCl, pH 7.5, and 1 mM ethylenediaminetetraacetic acid) using the NEPA21 electroporator (Nepa Gene, Chiba, Japan). The parameters were as follows: voltage 250V; pulse length, 3 ms; pulse interval, 50 ms; number of pulses, 2; decay rate; 10%; polarity + as poring pulse and voltage, 20 V; pulse length, 50 ms; pulse interval, 50 ms; number of pulses, 5; decay rate; 40%; polarity +/- as transfer pulse. Subsequently, the cell suspension was diluted with fresh liquid KH medium and cultured at 26°C for 7 days for restoration.
Reverse transcription (RT)-PCR
Total RNA was prepared from wild-type and dsRNA-introduced Euglena cells using the RNAiso regent (TaKaRa, Shiga, Japan). The first strand cDNA was synthesized using a PrimeScript II 1st strand cDNA kit (TaKaRa) with an oligo(dT) primer. The PCR was performed using specific primers as listed in S1 Table. EF1-α from Euglena (X16890) was used as a normalization gene.
Recombinant TER preparation
The open reading frames of Euglena TER were amplified from the first strand cDNA pool using the primer sets as listed in S1 Table. The amplified DNA fragments were ligated into the pGEM-T easy vector (Promega, Madison, WI, USA) to confirm the absence of PCR errors. The resulting constructs were digested with suitable restriction enzymes and were ligated into the expression vector pCold II (TaKaRa) to produce His-tagged proteins. The resulting plasmids were introduced into Escherichia coli strain BL21 Star cells (Agilent Technologies, Santa Clara, CA, USA). The E. coli cells transformed with each construct were grown in 3 mL of Luria-Bertani (LB) medium containing 50 μg mL-1 ampicillin. After an overnight culture at 37°C, the cultures were transferred to 600 mL of LB medium (with ampicillin) and grown to an A600 of 0.5. Isopropyl-1-thio-β-D-galactopyranoside was added to a concentration of 0.5 mM and the cells were incubated for 20 h at 15°C. The harvested cells were resuspended in 100 mM potassium phosphate buffer, pH 7.0 and sonicated. His-tagged recombinant EgTER proteins were purified using a TALON Metal Affinity resin (Clontech, Mountain View, CA, USA) according to the manufacturer’s instructions. Protein contents were determined using Bradford reagents. The purified enzymes were then desalted and concentrated using an ultrafiltration membrane (Amicon Ultra-4, Millipore, Billerica, MA, USA), and stored at −20°C until use.
TER activity measurement
TER activity was measured as described previously . The reaction mixture contained 100 mM potassium phosphate buffer, pH 6.2, 0.4 mM NADPH, 0.5 mM crotonyl-CoA, and enzyme. The oxidation of NADPH was followed by a decrease in absorbance at 340 nm (ε = 6.22 mM-1 cm-1). The protein concentration of enzyme solution was measured using the Coomassie Blue protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA).
Determination of chlorophyll
For extraction of chlorophyll, Euglena cells harvested from 1 mL of culture were vortexed in 1 mL of 80% (v/v) acetone. After centrifugation at 10,000 g for 5 min, chlorophyll in the supernatant was measured as described by Arnon .
Wax ester measurement
Extraction of the total wax ester fraction from Euglena cells was performed according to the method described by Inui et al . Euglena cells were harvested, freeze-dried, and added to 2.4 mL of a mixture of chloroform, methanol, and water in the ratio 10:20:8 (v/v/v). After thorough agitation, the mixture was centrifuged to remove cells and debris. The extraction was repeated, and the combined supernatants were added to 1 mL each of chloroform and water, followed by vigorous shaking. After centrifugation, the chloroform phase was evaporated and dissolved in hexane. The wax ester fraction was filtered using a polytetrafluoroethylene 0.22-μm filter for gas chromatography-mass spectrometry (GC-MS) analysis. The wax ester fraction was separated and determined using a GCMS-QP2010 instrument (Shimadzu, Kyoto, Japan). Separation was performed on an Agilent J&W DB-5ms column (30 m × 0.25 mm internal diameter, 0.25 μm film thickness; Agilent Technologies). A 1-μL portion of the wax ester fraction was injected into GC-MS using a splitless injection; helium was used as the carrier gas (1.16 mL/min). Chromatographic separation was initially set at 100°C (1 min), then the temperature was increased to 280°C (10°C per min) and held for 10 min. The mass transfer line and ion source were at 250°C. Myristyl myristate was detected using electron ionization (70 eV) in the selected ion monitoring mode at m/z 229.2 and 57.1 for the quantitative analysis.
Fatty acids measurement
Euglena cells were harvested, freeze-dried, and then the whole fatty acids were methyl esterified using a Fatty Acid Methylation kit and FAME purification kit (Nacalai Tesque, Kyoto, Japan). The purified FAME was analyzed by GC-MS with the condition described above by using the FAME compound library supplied by the manufacture.
Paramylon contents were determined using the phenol-sulfuric acid method with glucose solution as a standard as previously described by Tanaka et al .
Approximately 107 cells were collected from three individual cultures of aerobic and anaerobic-treated Euglena cells for analysis. Anaerobic treatment was performed as follows: a 5 mL of aliquot of the aerobic cell culture was dispensed into 15 mL plastic tube and bubbled with N2 gas for 1 min. Tube were then tightly capped and allowed to stand by shading for 24 h. The metabolite was extracted by chloroform–methanol–water at 1:1:0.4 (v/v) as previously described . The capillary electrophoresis–time-of-flight mass spectrometry (CE–TOFMS) and liquid chromatography (LC)–TOFMS conditions for cationic and anionic metabolite, and lipid analyses were as described elsewhere [20–23]. Raw data from the TOFMS analyses were processed using MasterHands software [21–23]. For the CE–TOFMS analysis, all the compounds, including cationic and anionic compounds, were identified by matching them to standard compounds in the Keio University’s library. For the LC–TOFMS analysis, several major compounds were also identified in this manner, with the others identified based on theoretical m/z values with mass accuracy of 20 ppm and orderly shift of retention time, described by Ikeda et al . Quantities were calculated using an internal standard: methionine sulfone for cationic metabolites, D-camphor-10-sulfonic acid for anionic metabolites, and dihexanoyl (d22)-sn-glycero-3-phosphocholine for lipids analyses. The position of double bonds or chirality was not identified in this study. All peaks were visually confirmed.
Generation and characterization of EgTER1 knockdown cells
To study the physiological significance of EgTER1, its knockdown cells (KD-ter1) were generated through electroporation-mediated dsRNA introduction based on established procedures [5,18]. Notably, when grown under heterotrophic conditions, the dsRNA-introduced cells exhibited a severe bleaching phenotype with a deficiency in chlorophyll (Fig 2E and 2F). Semi-quantitative RT-PCR and protein blotting confirmed that EgTER1 expression was markedly reduced in the knockdown cells compared to that in the mock control (Fig 2A and 2B). This resulted in an approximately 90% reduction in total TER activity against crotonyl-CoA (Fig 2C), indicating that EgTER1 constitutes a major isoform that recognizes a short chain CoA like crotonyl-CoA.
(a) RT-PCR for verification of EgTER1 gene knockdown. RT-PCR was carried out using total RNA from Euglena cells in which dsRNA was introduced. Mock cells were electroporated without dsRNA. (b) Immuno detection of TER1 protein. (c) Comparison of TER activity between mock control and KD-ter1 cells. Euglena cells grown to stationary phase were collected for TER activity measurement as described in the Material and Method section. An asterisk denotes statistically significant differences (*P<0.05) compared with the mock control. Values represent the means ± SD of three independent experiments. (d) Growth curve of mock control (blue) and KD-ter1 (orange) cells. The cultures were incubated in the heterotrophic KH medium. (e) Green color-less phenotype of KD-ter1 cells. The picture shows a representative culture after 7 days growth in the KH medium. (f) Influence of EgTER1-knockdown on chlorophyll content. An asterisk denotes statistically significant differences (**P< 0.01) compared with the mock control. Values represent the means ± SD of three independent experiments. (g) Effect of gene expression levels of other TER orthologs on EgTER1-knockdown. RT-PCR was carried out using total RNA from Euglena cells in which dsRNA was introduced.
Despite its bleaching phenotype, growth of KD-ter1 was indistinguishable from that of mock control cells under the heterotrophic conditions (Fig 2D). Thus, the colorless phenotype was not due to any apparent cellular damage caused by EgTER1 knockdown. Conversely, when grown under autotrophic conditions (without sugar supplementation), an obvious growth retardation of KD-ter1 cells was observed. However, its bleaching phenotype was considerably restored under these conditions (S1 Fig). This might have occurred because cells with a low RNAi effect may have preferentially grown under the conditions provided following dsRNA introduction.
To address whether the bleaching phenotype was due to knockdown of the EgTER1 gene in more detail, knockdown experiments were carried out using other regions of the EgTER1 sequence as triggers for RNAi (S2 Fig). In all cases, greening was impaired in KD-ter1 cells with reduced transcript levels of EgTER1. These results indicated that EgTER1 is required for the greening process in Euglena. It should be noted that the EgTER1 knockdown did not affect the expression of other potential TER genes (i.e., EgTER2-5, see the last subsection of ‘Results‘) indicated from our previous RNA-Seq analysis  (Fig 2G).
Wax ester fermentation in EgTER1 knockdown cells
Next, wax ester production in KD-ter1 cells was evaluated. Considering its impact on cell growth, the following analyses were performed using Euglena cells grown under heterotrophic conditions unless otherwise stated. Control and KD-ter1 cells grown under heterotrophic conditions with air were transferred to anaerobic conditions. In response to this treatment, levels of myristyl myristate (C28), a major component of Euglena wax esters, were markedly increased in both control and knockdown cells (Fig 3A). However, the accumulation was slightly albeit more pronounced in KD-ter1 cells compared to that in the control. Moreover, even prior to anaerobic treatment, myristyl myristate accumulation was higher in KD-ter1 cells. A comparable result was observed when EgTER1 was knocked down using different RNAi trigger regions (S2 Fig). Levels of methyl myristate (C14), a precursor of C28 wax ester, were also significantly higher in KD-ter1 cells, although knockdown of EgTER1 did not affect other fatty acids (C16 and C18) (Fig 3C). A fatty acid profile analysis also indicated that the amount of fatty acids with the carbon length ranging from C12 to C15 increased equally, clearly supporting the results of the wax ester accumulation in TER1 silencing cells (S3 Fig). These observations were unlikely to result from compensation by other potential TER enzyme(s), because KD-ter1 cells retained a low activity toward crotonyl-CoA even during anaerobic stress (Fig 3D). Conversely, paramylon accumulation and degradation were not affected by the EgTER1 knockdown under aerobic conditions and subsequent anaerobic treatment (Fig 3B).
(a) Influence of EgTER1-knockdown on myristyl myristate production under anaerobic conditions. Euglena cells grown to stationary phase were anaerobically treated for 24 h and then collected for wax ester measurement as described in the Material and Method section. The inset shows the results for 0 h in detail. An asterisk denotes statistically significant differences (*P<0.05, **P< 0.01) compared with the mock control. Values represent the means ± SD of three independent experiments. (b) Paramylon contents in EgTER1-knockdown cells. Cells grown to stationary phase were anaerobically treated for 24 h and collected for paramylon measurement. Values represents the means ± SD of three independent experiments. (c) Fatty acid content. Cells grown to stationary phase were anaerobically treated for 24 h and collected for fatty acids measurement. Values represent the means ± SD of three independent experiments. An asterisk denotes statistically significant differences (*P<0.05) compared with the mock control. (d) TER activity prior to and following anaerobic treatment. Values represent the means ± SD of three independent experiments.
Because KD-ter1 cells were severely bleached, the marked wax accumulation may have occurred because of a secondary effect of the loss of functional chloroplasts. To address this issue, the same knockdown assay was performed on a Euglena strain lacking chloroplasts (SM-ZK). As a result, the accumulation of myristyl myristate was again more pronounced in the KD-ter1 cells than that in the control under anaerobic conditions. Thus, our findings indicated that Euglena could produce wax esters without EgTER1 activity (i.e., the crotonyl-CoA reduction activity).
Metabolomic profile of TER1 knockdown cells
Metabolome analysis was next performed using KD-ter1 cells prior to (0 h) and following (24 h) anaerobic treatment to determine the further implications for the effect of EgTER1 on metabolism. This encompassed some kinds of lipids, sugars, amino acids, and ATP. The enhanced production of wax ester in KD-ter1 cells was also observed in this metabolome analysis (Fig 4). However, glycolipids, such as monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylglycerol (PG), all of which are components of chloroplast membranes, were poorly detected in KD-ter1 cells. This was consistent with the colorless phenotype of the knockdown cells. Levels of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), being glycerophospholipids, were higher in the knockdown cells compared to the control both before and after anaerobic treatment. As the PC is a precursor of chloroplastic glycolipids, MGDG, DGDG and SQDG, it is reasonable to conclude that the higher accumulation of both PC and PE occurs as a result of the suppression of these biosynthesis in the knockdown cells. In the control cells, ATP levels were markedly decreased upon anaerobic treatment, which was also the case in KD-ter1 cells. Conversaly, ATP levels in KD-ter1 cells were much higher than those in the control cells both prior to and following anaerobic treatment. As shown in Table 1, the knockdown of EgTER1 had a large effect on the levels of amino acids. For example, in KD-ter1 cells, the levels of alanine, glycine, tryptophan, and arginine were highly accumulated under aerobic conditions, then significantly decreased upon anaerobic treatment. Thus, the EgTER1 knockdown perturbed several metabolic pathways, which might result in the notable accumulation of wax ester.
Concentrations of some key metabolites related to the glycolysis (a), TCA cycle (c), storage lipid (c) and membrane lipid (d) metabolism. Cells grown to stationary phase were anaerobically treated for 24 h and collected for metabolome analysis. Concentrations and relative amounts of each metabolite in the mock control and EgTER1-knockdown cells are shown. An asterisk denotes statistically significant differences (*P<0.05, **P< 0.01) compared with the mock control. Values represent the means ± SD of three independent experiments.
Other potential TER genes
Our recent RNA-Seq data  showed that there are five genes annotated as TER; comp34527_c0_seq1_5 (EgTER1), comp27719_c0_seq1_4 (EgTER2), comp22878_c0_seq1_1 (EgTER3), comp27434_c0_seq1_3 (EgTER4), and comp32876_c0_seq1_1 (EgTER5). The sequence of EgTER1 amino acids is identical to that of the TER enzyme identified by Hoffmeister et al . Amino acid sequences of these enzymes show identity (ranging from 6.8 to 29.1%) and similarity (from 45.3 to 73.8%) to each other. A domain search using the Pfam database (http://pfam.xfam.org/, ) showed that only EgTER1 has an obvious Enoyl-CoA_reductase domain (PF12241), which is located between the NAD(P)H- and FAD-domains (S4 Fig). This analysis found some domains related to alcohol dehydrogenase in EgTER4 and 5, but failed to find any domain in EgTER2 and 3 (S4 Fig). Although EgTER1 has an N-terminal extension that contains two potential transmembrane domains (S4 Fig), which is a characteristic of Euglena plastidic proteins , this enzyme was purified from Euglena mitochondria, demonstrating its mitochondrial localization. Although the prediction of Euglena protein localization using protein localization prediction tools is generally difficult, two such tools, Target P and WoLF PSORT, predicted that EgTER2, EgTER3, and EgTER5 are mitochondrial, whereas EgTER4 is cytosolic (S2 Table).
To investigate whether these potential TERs could catalyze the reduction of short chain length substrates, such as crotonyl-CoA, we attempted to generate their recombinant proteins. All EgTER proteins, except for EgTER3, could be expressed in and purified from E. coli cells. Consistent with previous report , EgTER1 exhibited substantial crotonyl-CoA reductase activity (3.20 ± 0.35 μmol/min/mg protein). In contrast, other enzymes did not show any remarkable activity with crotonyl-CoA as a substrate, although we did not check whether these enzymes could reduce other potential substrates.
We also tested the effects of knockdown of these genes on wax ester production. KD-ter2 cells could not grow even under heterotrophic conditions. In comparison, other knockdown cells (KD-ter3, 4 and 5) grew normally but, as with KD-ter1, they accumulated less chlorophyll compared to the mock control under heterotrophic conditions (Fig 5B). Knockdown of these genes had no negative impact on wax ester production under anaerobic conditions (Fig 5C). These findings suggested that these enzymes do not act as crotonyl-CoA reductases in the wax ester fermentation process.
(a) RT-PCR for verification of each possible TER gene knockdown. RT-PCR was carried out using total RNA from Euglena cells in which dsRNA was introduced. (b) Chlorophyll content in possible TER gene knockdown cells. An asterisk denotes statistically significant differences (*P<0.05) compared with the mock control. Values represent the means ± SD of three independent experiments. (c) Wax ester production under anaerobic conditions. Euglena cells grown to stationary phase were anaerobically treated for 24 h and collected for myristyl myristate measurement as described in the Material and Method section. Values represent the means ± SD of three independent experiments.
An outline of wax ester fermentation in Euglena upon anaerobic treatment was proposed by Inui et al . At that time, it was still unclear how this process leads to the net gain of ATP, because only fatty acid synthesis pathways that require malonyl-CoA were known to operate in Euglena. That is, if these ATP-dependent pathways were involved in the wax ester production, the consumption of ATP was expected to exceed its generation in glycolysis. Soon thereafter, observation of TER activity against short chain length substrates (such as crotonyl-CoA) in mitochondria led to the proposal of a novel mitochondrial pathway for fatty acid synthesis that does not require ATP consumption, providing an explanation for the net gain of ATP in wax ester fermentation . As previously noted, TER was strongly expected to constitute the rate-limiting enzyme in the mitochondrial pathway. The purpose of the present study was therefore to provide further evidence for the physiological role of TER in the fermentation process: however, no positive answer was provided from our reverse genetic analysis.
EgTER1 is identical to the enzyme cloned by Hoffmeister et al . In the present study, approximately 90% reduction in total crotonyl-CoA reductase activity was found in KD-ter1 cells, demonstrating the enzyme to be a major isoform. Nevertheless, knockdown of EgTER1 had no negative impact on wax ester production under anaerobic conditions. On the contrary, wax ester production was more pronounced in the knockdown cells as compared to that in the mock control. These findings demonstrated that EgTER1 is dispensable for wax ester production in E. gracilis under anaerobic conditions. However, the mechanism by which KD-ter1 cells produced wax esters remains unknown. Judging from the primary structures and enzymological properties, other potential EgTER genes were unlikely to encode a crotonyl-CoA reductase. In comparison, Inui et al  purified an NADH-specific crotonyl-CoA reductase from Euglena mitochondria, although this has not yet been cloned. This isoform consisted of 15 and 25 kDa subunits and differed substantively from EgTER1, which has a molecular mass of 44 kDa. This enzyme might therefore be a component of the residual (approximately 10%) crotonyl-CoA reductase activity in KD-ter1 cells. However, it should be noted that TER activity in Euglena is extremely low compared to the activity of the other enzymes in the mitochondrial fatty acid synthesis pathway [8,12–14]. Accordingly, the residual 10% crotonyl-CoA reductase activity that was retained in KD-ter1 cells was unlikely to be sufficient to produce wax esters. Moreover, the residual activity cannot explain the pronounced wax ester synthesis in the knockdown cells. Alternative pathway(s) must therefore exist to substitute for the wax ester production in the absence of EgTER1. As shown by our metabolome analysis, large metabolic perturbations occurred in KD-ter1 cells, which might result in the formation of new metabolic route(s) for fatty acid synthesis that might not be operating in the wild-type organism. Notably, genes involved in fatty acid biosynthesis were found to be upregulated in response to anaerobic conditions in our previous RNA-Seq analysis . The increase in ketogenic amino acids like Leu, Ile, and Trp may be attributed to this phenomenon. Together, these observations suggest the existence of an alternative route for anaerobic wax ester synthesis. Recently, Nakazawa et al.,  have reported that fatty acids are anaerobically synthesized in Euglena mitochondria by the reversal of β-oxidation, where trans-2-enoyl-CoA is reduced by acyl-CoA dehydrogenase (ACD1) using the electrons provided by NADH via the electron transport chain complex I, rhodoquinone, and electron transfer flavoprotein. Thus the reversal reaction of ACD1 would be one of the strong alternative possibilities. However, the contribution of ACD1 still admits of further argument to reach a crucial conclusion, because the ACD1 gene silencing cells show approximately 60% of residual accumulation of wax esters .
Another notable finding was that KD-ter1 showed a bleaching phenotype concomitant with negligible levels of chlorophylls and glycolipids, indicating a critical role of EgTER1 for greening process (chloroplast development and/or chlorophyll biosynthesis). However, the role of this enzyme in greening is currently unknown. It is unlikely that the chlorophyll-less phenotype was due to cellular damage caused by EgTER1 silencing, because the knockdown cells grew similar to the mock control cells level under heterotrophic conditions. Alternatively, EgTER1 contains two transmembrane helices in its N-terminal region, which is a typical property of chloroplast-targeted proteins in Euglena . Together with this characteristic, the high similarity between EgTER1 and FabV (that is enoyl-acyl-ACP reductase) sequences allows the speculation that EgTER1 might represent a candidate for enoyl-acyl-ACP reductase activity, as-yet unidentified, in the chloroplastic FAS II pathway. Although Hoffmeister et al  failed to detect the NADH-dependent crotonyl-CoA reductase activity in chloroplasts purified from Euglena cells, this is not decisive evidence that EgTER1 is a non-chloroplastic enzyme. Further analysis will be required to clarifying whether this enzyme can function in the FAS II system for chloroplast development.
Taken together, these findings indicate that EgTER1 constitutes a major isoform of crotonyl-CoA reductases, albeit is dispensable for wax ester production under anaerobic conditions. How Euglena produces wax esters without TER activity is currently unknown. Further detailed biochemical and metabolomic analyses combined with reverse genetics will be required to answer this question in future studies.
S1 Fig. Effect of EgTER1-knockdown on cell growth and chlororphyll content in autotrophically grown cells.
(a) RT-PCR for verification of EgTER1 gene knockdown. RT-PCR was carried out using total RNA from Euglena cells in which dsRNA was introduced. Mock cells electroporated without dsRNA. (b) Green color-less phenotype of KD-ter1 cells. The picture shows a representative culture after 15 days growth in the autotrophic CM medium. (c) Growth curve of mock control (blue) and KD-ter1 (orange) cells. The cultures were incubated in the autotrophic CM medium. An asterisk denotes statistically significant differences (*P< 0.05) compared with the mock control. Values represent the means ± SD of three independent experiments. (d) Total chlorophyll content. An asterisk denotes statistically significant differences (*P< 0.05) compared with the mock control. Values represent the means ± SD of three independent experiments.
S2 Fig. Confirmation of the bleaching phenotype of EgTER1-knockdown cells by using other regions of EgTER1 sequence as triggers for RNAi.
(a) EgTER mRNA organization and positions of primers used for the gene knockdown. Each arrowhead pair with different colors indicates the regions where individual dsRNAs were generated. (b) RT-PCR for verification of EgTER1 gene knockdown. (c) Green color-less phenotype of KD-ter1 cells. The picture shows a representative culture after 7 days growth in the heterotrophic KH medium. (d) Total chlorophyll content. An asterisk denotes statistically significant differences (*P< 0.05, **P< 0.01) compared with the mock control. Values represent the means ± SD of three independent experiments. (e) Myristyl myristate content. Euglena cells grown to stationary phase were anaerobically treated for 24 h and collected for wax ester measurement. An asterisk denotes statistically significant differences (*P< 0.05) compared with the mock control. Values represent the means ± SD of three independent experiments.
S3 Fig. Comparison of fatty acids profile in mock and EgTER1-knockdown cells.
Euglena cells grown to stationary phase were anaerobically treated for 24 h and then collected for fatty acids measurement as described in the Material and Method section.
S4 Fig. Putative transmembrane domains predicted by the TMHMM program (http://www.cbs.dtu.dk/services/TMHMM/) and putative motif domains indicated by the Pfam database (http://pfam.xfam.org/).
The top picture shows the predicted known motifs in each EgTER. The striped profile in the bottom picture shows the probability for TM helix.
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