Plant Biotechnology Journal (2015), pp. 1–10
doi: 10.1111/pbi.12398
Elevated CO2 improves lipid accumulation by increasing carbon metabolism in Chlorella sorokiniana Zhilan Sun, Yi-Feng Chen and Jianchang Du* Institute of Biotechnology, Provincial Key Laboratory of Agrobiology, Jiangsu Academy of Agricultural Sciences, Nanjing, China
Received 27 January 2015; revised 14 April 2015; accepted 16 April 2015. *Correspondence (Tel +86-025-84392767; fax +86-025-84392767; email [email protected])
Keywords: Chlorella sorokiniana, photosynthesis, carbon dioxide, transcriptomics, lipid, carbon flow.
Summary Supplying microalgae with extra CO2 is a promising means for improving lipid production. The molecular mechanisms involved in lipid accumulation under conditions of elevated CO2, however, remain to be fully elucidated. To understand how elevated CO2 improves lipid production, we performed sequencing of Chlorella sorokiniana LS-2 cellular transcripts during growth and compared transcriptional dynamics of genes involved in carbon flow from CO2 to triacylglycerol. These analyses identified the majority genes of carbohydrate metabolism and lipid biosynthesis pathways in C. sorokiniana LS-2. Under high doses of CO2, despite down-regulation of most de novo fatty acid biosynthesis genes, genes involved in carbohydrate metabolic pathways including carbon fixation, chloroplastic glycolysis, components of the pyruvate dehydrogenase complex (PDHC) and chloroplastic membrane transporters were upexpressed at the prolonged lipid accumulation phase. The data indicate that lipid production is largely independent of de novo fatty acid synthesis. Elevated CO2 might push cells to channel photosynthetic carbon precursors into fatty acid synthesis pathways, resulting in an increase of overall triacylglycerol generation. In support of this notion, genes involved in triacylglycerol biosynthesis were substantially up-regulated. Thus, elevated CO2 may influence regulatory dynamics and result in increased carbon flow to triacylglycerol, thereby providing a feasible approach to increase lipid production in microalgae.
Introduction Nutrient limitation (deprivation of nitrogen or phosphorus) is the primary method used to enhance lipid production in microalgae (Yang et al., 2013). For example, the lipid content of Chlorella vulgaris grown under nitrogen deprivation was estimated to be up to 70% (Amaro et al., 2011). Likewise, when subjected to nitrogen or phosphorus limitation, Scenedesmus sp. showed an increase in lipids as high as 30% and 53%, respectively (Xin et al., 2010). It is widely accepted that when there is insufficient nitrogen for protein synthesis, excess carbon from photosynthesis is channelled into storage molecules such as triacylglycerols (Griffiths et al., 2014). In general, this would limit cell growth while increasing lipid content (Mata et al., 2010). Therefore, a two-stage process was proposed for photosynthetic microalgae. In the first stage of this process, cells are first grown under nutrient sufficient conditions to obtain high biomass accumulation. In the second stage of this process, cells are grown under nutrient limited conditions to enhance lipid synthesis (Rodolfi et al., 2009). The mechanism of lipid accumulation under nutrient limitation may be that it provides cells excess carbon by depriving nitrogen (Li et al., 2014; Valenzuela et al., 2012). However, this stress condition would limit the growth of microalgal cells (Mata et al., 2010). An alternative strategy would be to provide excess carbon in the form of carbon dioxide (CO2), whereby growth and lipid accumulation might be achieved in a so-called one-stage process (Cheng et al., 2013). Extra CO2 supply is believed to be a more
promising approach for scale-up lipid production than nitrogen deprivation because microalgae may assimilate CO2 from the flue gas of factories, thereby upgrading this waste stream into usable products such as lipid (Reijnders, 2013; Shen, 2014). Microalgae are so far the only species known to be able to utilize extremely high levels of CO2 up to 20% (Solovchenko and KhozinGoldberg, 2013). For example, Botryococcus braunii could grow well under 20% CO2 even without any adjustment of culture pH (Ge et al., 2011). Scenedesmus sp. and Chlorella sp. were also reported to grow well when exposed to a gas mixture containing up to 20% CO2 (Westerhoff et al., 2010). However, the majority of previous studies have focused on the effects of medium composition, illumination strategies, or various photo-bioreactor setups on the growth and lipid production of microalgae; the mechanism of lipid accumulation under high doses of CO2 has not been fully elucidated (Pires et al., 2012). Valenzuela suggested that the build-up of precursors to the acetyl coenzyme A (acetyl-CoA) carboxylases may play a more significant role in triacylglycerol (TAG) synthesis rather than the actual enzyme levels of acetyl-CoA carboxylases under nitrogen deprivation conditions (Valenzuela et al., 2012). They hypothesized that carbon was being ‘pushed’ into fatty acid (FA) synthesis via elevated acetyl-CoA and NADPH levels and thus not being ‘pulled’ by a large abundance of FA synthesis activity (Valenzuela et al., 2012). Our previous work revealed high carbon–lipid conversion efficiencies in a high-dose CO2 culture system when compared with cultures grown under ambient CO2 (air) conditions. This suggests that more fixed carbon might flow into the
Please cite this article as: Sun, Z., Chen, Y.-F. and Du, J. (2015) Elevated CO2 improves lipid accumulation by increasing carbon metabolism in Chlorella sorokiniana. Plant Biotechnol. J., doi: 10.1111/pbi.12398
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2 Zhilan Sun et al. lipid synthesis pathway under high doses of CO2. Based on this, we proposed that carbon flow may also be driven by increasing the flux of CO2 to acetyl-CoA in microalgal chloroplasts in response to high doses of CO2 leading to increased lipid accumulation. To clarify how carbon was channelled into lipid synthesis pathways, we employed Illumina paired-end sequencing of C. sorokiniana cellular transcripts collected under variable growth conditions. Global transcriptome analysis revealed a detailed carbon metabolism pathway from CO2 to acetyl-CoA, which is the key precursor for FA biosynthesis in chloroplasts. Furthermore, transcriptional dynamics of acetyl-CoA producing associated genes provided evidence that enhanced carbon flow from CO2 to acetyl-CoA in the chloroplast might be the critical factor for lipid accumulation under high doses of CO2. Additionally, transcriptional dynamics analysis revealed that TAG synthesis-associated genes could be another factor involved in enhanced lipid accumulation. These findings improve our understanding of lipid production in microalgae under high doses of CO2 and will provide a new platform for future genetic engineering in microalgae for the overproduction of lipid.
carbohydrate metabolism (319), amino acid metabolism (257), cofactor and vitamin metabolism (189), and lipid metabolism (159) (Figure 3). Pathways related to translation, organismal systems, cellular processes and environmental information processing were also well represented by unigenes from C. sorokiniana LS-2. These data provided a valuable resource for investigating metabolic pathways in C. sorokiniana LS-2 cells when grown under air or CO2 aeration. Of the global metabolic pathway, our particular attention would focus on the energy and substance metabolism including photosynthesis, glycolysis, tricarboxylic acid cycle (TCA cycle), FA and TAG biosynthesis because C. sorokiniana LS-2 had the potential to accumulate lipid under high doses of CO2. Additionally, the assigned photosynthesis pathway suggested that C. sorokiniana possessed some unique features for the C4-like photosynthesis pathway. The C4 pathway is believed to improve photosynthetic carboxylation and reduce photorespiration via increasing CO2 flux to ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Therefore, the regulation of C4-like pathway response to high doses of CO2 was also investigated below.
A functional C4-like pathway exists in C. Sorokiniana
Results Sequencing, de novo assembly, and functional annotations To investigate the potential metabolic pathways involved in enhanced lipid synthesis under high doses of CO2, two sequencing libraries at the exponential phases were prepared from C. sorokiniana LS-2 and sequenced with the Illumina paired-end HiSeq platform. Growth of LS-2 under air and 10% CO2 aeration was monitored by biomass measurement (Figure S1). There were 125,162,732 clean reads generated from C. sorokiniana LS-2. Of the clean reads, 94.96% bases had a Q-value ≥20. All the raw data have been submitted to SRA database (SRP056572). De novo assembly generated 22 432 unigenes with an average length of 977 bp and N50 of 1680 bp. This Transcriptome Shotgun Assembly project has been deposited at DDBJ/EMBL/ GenBank under the Accession Number GCUV00000000. The version described in this study is the first version, GCUV01000000. Of these 22 432 unigenes, 45.52% were annotated with GO terms. They were classified into 3 functional categories: molecular function, biological process and cellular component (Figure 1). The matched unique sequences assigned to molecular function were clustered into 12 classification bins with the largest subcategory involved in ‘binding’ (68.35%) and the second largest subcategory involved in ‘catalytic activity’ (21.35%). The unique sequences were classified into 20 bins with the most abundant bin comprising transcripts involved in metabolic (53.5%) and cellular processes (20.9%). In terms of cellular components, the unique sequences were divided into 14 classifications with the most abundant being classified as ‘cell part’ (30.0%) and ‘cell’ (19.7%). The 5163 matched unique sequences were divided into 26 categories, using KOG proteins (Figure 2). The dominant category includes general functional prediction, post-translational modification, protein turnover and chaperon, and signal transduction, respectively. For further detailed understanding of their function, unique sequences were then analyzed for KO identifiers using the KEGG database. Of the 5163 matched unique sequences, 4666 of the KO annotated unigenes were capable of being assigned to KEGG biochemical pathways. The most well-represented metabolic pathways are involved in energy metabolism (329),
Rubisco is the central carboxylation enzyme for CO2 fixation during photosynthesis, but its affinity for CO2 is low (Giordano et al., 2005). To solve the problem, several microalgae have developed a C4-like CO2 concentration mechanism (CCM) to maintain photosynthetic carboxylation when cells encounter low CO2 availability (Chang et al., 2013; Haimovich-Dayan et al., 2013). In the C4-like pathway, relatively high affinity carboxylases fix inorganic carbon into C4 compounds that can be transported to the chloroplast. The C4 intermediates are then decarboxylated to deliver the inorganic carbon to the relatively low-affinity Rubisco enzyme (Worden et al., 2009). Until now, the C4-like pathway has been identified in Phaeodactylum tricornutum (Kroth et al., 2008), Thalassiosira pseudonana (Reinfelder et al., 2004), Ostreococcus tauri (Derelle et al., 2006), Myrmecia incisa and Micromonassp (Ouyang et al., 2013; Worden et al., 2009). However, no similar pathway was reported in Chlorella. According to the transcriptome analysis, our data showed that a C4-like photosynthesis might exist in C. sorokiniana LS-2 (Figure 4 and Table S1). C. sorokiniana appears to encode the enzymes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) and pyruvate orthophosphate dikinase (PPDK, EC 2.7.9.1). Each of these enzymes is necessary for C4-metabolism. A unique sequence (comp7826_c0) encoding PEPC has been identified. The signal and transit peptide were not found in this PEPC according to subcellular localization analysis, indicating that it might function within the cytosol to produce oxaloacetic acid (OAA). Subsequently, OAA may be transferred to the mitochondria or chloroplast for further decarboxylation. On the one hand, OAA was decarboxylated to pyruvate by malate dehydrogenase (MDH) and the downstream malic enzyme (ME). Three unique sequences encoding MDHNAD+ (comp10051_c0 and comp10035_c0) and ME-NAD+ (comp7698_c0) were identified, and they possessed a predicted mitochondrial transit peptide, respectively, according to subcellular localization analysis. OAA could also be decarboxylated to phosphoenolpyruvate (PEP) by a mitochondria-localized PEPCK. Two unique sequences (comp3474_c0 and comp9353_c0) were identified to encode PEPCK, including one that had 71% amino acid identity with Auxenochlorella protothecoides [GenBank:
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Mechanism of lipid accumulation under high-dose CO2 3
Figure 1 Gene ontology classification of assembled unigenes. The 22, 432 matched unigenes were classified into 3 functional categories: molecular function, biological process and cellular component.
could be carboxylated to OAA again by a pyruvate carboxylase (PYC) in mitochondria. Subsequently, the resulting OAA could participate in the TCA cycle. On the other hand, both chloroplast-localized MDH-NADP+ and its downstream enzyme NADP+-dependent ME were also identified in C. sorokiniana LS-2. Therefore, the OAA is assumed to be reduced to pyruvate with the help of the two enzymes, which target the chloroplast. The product of the decarboxylation is CO2 which can be subsequently fixed by Rubisco. Afterwards, the pyruvate may be converted by PPDK to PEP, which is then transported by PEP/Pi translocators to the cytosol, where carboxylation is performed by PEPC. A unique sequence (comp3367_c0) encoding PPDK had been identified. The subcellular localization of PPDK is unknown due to the lack of N-terminal sequence; however, the identified PEP/Pi translocator in the chloroplast implies that there should be one chloroplastic PPDK in C. sorokiniana. In summary, the identification of key enzymes indicates that a functional C4 pathway might exist in C. sorokiniana. According to the localization of those crucial enzymes, it may have two pathways for CO2 production via decarboxylation of OAA and malate in the mitochondria and chloroplast. However, CO2 utilization by Rubisco only occurs in the chloroplast. The C4-like photosynthesis pathway has long been believed to enable the microalgae to adapt to a low CO2 level habitat. Additionally, the energetic cost of a futile cycle in mitochondria implied that the C4 metabolism may have a function under excess light energy. Those suggest that the flow of metabolites in this pathway would be affected by CO2 concentration and light intensity.
Mechanism of lipid accumulation with elevated CO2 supply Figure 2 KOG functional classification of all unigenes. A total of 5163 unigenes showed significant similarity to the sequences in KOG databases and were clustered into 26 categories.
KFM29076.1]. Moreover, this identified unique sequence revealed an OAA and an ATP-binding site (Trapani et al., 2001). Additionally, a unique sequence (comp3865_c1) encoding pyruvate carboxylase (PYC) was identified and it possessed a predicted mitochondrial transit peptide. Therefore, the generated pyruvate
To clarify the molecular mechanism of lipid accumulation under high doses of CO2, we compared patterns in global gene expression of exponentially grown cells of strain LS-2 under air and CO2 aeration via mRNA-Seq. The transcriptomes were validated by qPCR analysis of 61 selected genes, and the coefficient of determination (R2) between qPCR-based and mRNA-Seq-based transcript abundance was 0.802 (Figure S2). Most of the selected genes showed a positive correlation between mRNA-Seq and qPCR (Figure S2). The significant DEGs
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Figure 3 KEGG classification of assembled unigenes. The 4666 KO annotated unigenes were assigned to 5 KEGG biochemical pathways: metabolism, genetic information processing, organism system, cellular processes and environmental information processing.
Figure 4 Model of the C4-like pathway in C. sorokiniana LS-2 based on the transcriptome. The dashed line indicates the route that was not detected in this transcriptome.
(different expression genes) were then identified with the applied criteria [q-value 1] (Figure 5). 1837 genes (8.2% of total) were found to be significantly upregulated, whereas 990 genes (4.4% of total) were downregulated in response to high CO2 aeration (Data S1). All DEGs were mapped to terms in the KEGG database. Because of the particular interest of this study in lipid synthesis under high-dose CO2 utilization, the genes involved in carbon fixation, carbohydrate metabolism and lipid synthesis were further studied (Table S1). A total of 87 differentially expressed genes related with carbon flow to lipid accumulation were identified. Among them, 51 genes (58.6%) were found to be significantly up-regulated, whereas 36 genes (37.2%) were down-regulated in response to high dose CO2 aeration. Fortynine up-regulated genes were particularly enriched in carbon fixation, carbohydrate metabolism, accounting for almost all of the deferentially expressed genes. However, down-regulated genes were mainly enriched in FA and TAG biosynthesis pathway. Of 13 differentially expressed genes in the two pathways, 11 genes were down-regulated. In particular acetyl-CoA carboxylase,
the first key enzyme involved in FA biosynthesis was downexpressed by 3.2- to 17.9-fold. TCA cycle genes were upexpressed (from 2.3- to 95.8-fold) under high-dose CO2 aeration conditions (Table S1). Levels of transcripts encoding the two potentially rate-limiting enzymes, citrate synthase and isocitrate dehydrogenase, in the TCA cycle increased 11.5- and 11.9-fold, respectively. To our knowledge, the glycolysis generated acetyl-CoA can be broken down into CO2 via the TCA cycle to produce NADH that fuels ATP synthesis by oxidative phosphorylation in the mitochondria (Sweetlove et al., 2010). Moreover, the intermediates of the TCA cycle (e.g. 2-oxoglutarate, succinyl CoA, fumarate and oxaloacetate) are linked to amino acid metabolism (Li et al., 2014). Thus, increases in carbon flow through the TCA cycle may supply the assimilatory power for carbon fixation and lipid biosynthesis, and also increase the carbon flow into these TCA cycle intermediates for amino acid biosynthesis. Besides, malate, an intermediate of the TCA cycle, can be converted to pyruvate by a NAD+-malic enzyme (ME). Pyruvate, the key precursor for acetyl-CoA can subsequently be transported to chloroplast for FA biosynthesis.
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Mechanism of lipid accumulation under high-dose CO2 5 pyruvic acid (decarboxylate of MDH and ME) is an important precursor of the TCA cycle. Therefore, we speculate that the C4-like pathway might be more likely to function in replenishing TCA cycle intermediates for energy production as opposed to functioning to redirect CO2 into carbon fixation in the chloroplast. Additionally, the transcriptional dynamics of genes involved in the Calvin cycle were also examined by qPCR. They were upexpressed at P2 and P3 in the CO2 treatment with the exception of FBP, which exhibited similar expression in the CO2 and air treatments (Figures 6 and 7).
Elevated CO2 enhances the chloroplast glycolytic pathway
Figure 5 Comparison of expression patterns of differentially expressed unigenes identified between high-dose CO2 and air aeration. The red dots represent DEGs, and the blue dots represent non-DEGs.
Therefore, the up-regulation of the TCA reactions, coupled with the increased expression of genes involved in carbon fixation, may suggest that enhanced availability of carbon skeletons and reducing power for FA and TAG synthesis can be influenced by the availability of CO2. Taken together, these results could be consistent with the previous work that carbon is being ‘pushed’ into FA synthesis via elevated acetyl-CoA and NADPH levels and thus not being ‘pulled’ by a large abundance of FA synthesis activity (Valenzuela et al., 2012). Regulation of the genes involved in carbon flow from CO2 to lipid accumulation will be discussed in detail below.
Carbon precursor supply for FA synthesis was enhanced by high-dose CO2 Carbon fixation was enhanced by high-dose CO2 Transcriptomic analysis indicated that CO2 assimilation was slightly enhanced under the high-dose CO2 condition. Most genes in the C4-like pathway and Calvin cycle were up-regulated during exponential phase growth (Table S1). To further examine the transcriptional dynamics of those genes, qPCR was performed at the P1, P2 and P3 points of the growth cycle (Figure S1 and Table S2). According to NormFinder analysis, the stability value of candidate reference gene Actin, 18S rRNA, and Tubulin was 0.013, 0.037 and 0.041, respectively, indicating that they all exhibited the relative stability in expression level. To facilitate our further analysis, Actin was used as a reference gene. Under highdose CO2 aeration, two key enzymes PEPC and PPDK in C4-like pathway were significantly upexpressed at P1 and P2, however, both of them returned to the basal level at P3 (Figures 6 and 7). The data suggest that the C4-like pathway was active during cell growth phase (P1 and P2), but played a less important role during prolonged lipid accumulation (P3). Interestingly, mitochondrial MDH and ME were upexpressed at P2 and P3; however, no similar regulation was observed in chloroplast MDH and ME. Both of these enzymes are involved in CO2 formation and consider that
Glyceraldehyde-3-phosphate (G3P), an intermediate product of the Calvin cycle, is converted to pyruvate via glycolysis. The pyruvate is then converted to acetyl-CoA, the precursor of de novo FA biosynthesis. In Nannochloropsis, glycolytic pathways operate in both the plastid and the cytosol, which produces pyruvate for FA biosynthesis while generating the high-energy compounds ATP and NADH (Li et al., 2014). All the components of the glycolytic pathway were identified in C. sorokiniana, and subcellular localization analysis of these components revealed that glycolysis is likely to take place both in the cytosol and chloroplast. The transcriptional dynamics of genes involved in glycolysis were examined by qPCR (Table S2, Figures 6 and 7). At P2, most of the glycolytic genes in the cytosol and chloroplast were upexpressed under the CO2 condition, suggesting a more active metabolic activity at P2 under high doses of CO2. As the cells went into stationary phase (P3), genes located in the chloroplast remained up-regulated under high-dose CO2. In contrast, PGK and one PGAM in cytosol were significantly down-regulated. This suggests that enhanced FA synthesis under high CO2 may be due to up-regulation of glycolysis in chloroplasts, which could supply more carbon precursors, ATP and reducing equivalents for FA synthesis when CO2 is not limiting. Additionally, genes encoding transporters that direct carbon precursors to FA biosynthesis in chloroplasts were also identified. Bile acid:Na+ symporter (BASS) mediates sodium-coupled pyruvate import into plastids (Furumoto et al., 2011). Three unique sequences (Table S1) encoding a putative BASS were identified in LS-2. In this pathway, the balance of Na+ influx is maintained by a sodium:proton antiporter (NHD) (Li et al., 2014). Thirteen NHD isoforms were identified in LS-2. Transcriptional dynamics analyses revealed that selected BASS and NHD were up-regulated under high dose CO2 at P2 and P3. A putative phosphate/ phosphoenol pyruvate translocator (PPT), which imports cytosolic PEP into the plastid stroma in exchange for inorganic phosphate (Li et al., 2014), was identified and its transcript level also increased at P2 and P3. Therefore, the evidence collectively suggests that enhanced FA synthesis under high-dose CO2 was correlated with the up-regulation of transporter genes responsible for importing the intermediates (e.g. PEP and pyruvate) from the cytosol into the chloroplast.
High CO2 enhances biosynthesis of acetyl-CoA in chloroplast Similar to plant, in microalgae, palmitic acid (16:0) is believed to be synthesized de novo in the chloroplast from the precursor acetyl-CoA. There are two possible processes for supplying acetylCoA to the chloroplast. One mechanism is the direct catalysis of pyruvate by the plastid-localized pyruvate dehydrogenase complex (PDHC), which bridges the glycolysis and lipid biosynthesis pathways in the plastid. The other mechanism is an indirect regeneration from the imported cytosolic acetate (Oliver et al.,
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Figure 6 Transcriptional dynamics of individual genes related to the carbon flow from CO2 to TAG biosynthesis in response to high dose CO2 aeration. Relative fold differences were calculated based on the MCt method using the Actin amplification product as an internal standard.
2009). PDHC consists of three subunits: E1, E2 and E3. The plastidial PDHC genes were identified in LS-2 (Table S1), and transcriptional dynamics of E2 revealed that PDHC was significantly up-regulated at P2 and P3 (Table S2, Figures 6 and 7). Considering that lipid started to accumulate at P2, the remarkable increase of acetyl-CoA in the chloroplast at P2 and P3 might be the key factor that results in the ‘push’ of carbon into FA biosynthesis pathways by high-dose CO2. On the other hand, pyruvate can be converted to acetate by a pyruvate decarboxylase (PDC) and an aldehyde dehydrogenase (ALDH), which was called the ‘PDHC bypass’ pathway (Li et al., 2014), and then, the acetate can be transported to chloroplast where acetyl transfer was performed by acetyl-CoA synthetase. Two copies of PDC genes and three copies of ALDH genes located in the cytosol were found in LS-2. The transcriptional dynamics revealed that most of the selected genes were downexpressed in the presence of high dose CO2 (Figure 6 and 7), suggesting that the ‘PDHC bypass’ maybe not the major pathway of acetyl-CoA formation. Moreover, these data suggest that the chloroplast is the major site for acetyl-CoA formation in FA biosynthesis and not the cytosol.
Lipid accumulation is largely independent of de novo fatty acid synthesis The de novo synthesis of FAs begins with acetyl-CoA via the initial catalysis by acetyl-CoA carboxylase (ACCase). malonylCoA:acyl protein malonyltransferase (MCMT) subsequently transfer the malonyl moiety to an acyl-carrier protein (ACP)
to generate malonyl-ACP, which then enters into a series of carbon chain condensation reactions to form 16- or 18-carbon FAs. ACCase consists of four subunits encoded by accA, accB, accC and accD. Seven putative ACCase genes, two ACP genes and one copy of MCMT were identified in LS-2 (Table S1). Three selected ACCase genes as well as ACP and MCMT genes were down-regulated at P1, and subsequently, were upexpressed at P2, with expression only again declining at P3. None of these genes were upexpressed at the transcriptional level during prolonged lipid accumulation (P3). Similar results were recently reported in Phaeodactylum tricornutum, in which only modest changes in gene expression of FA metabolism genes were observed (Valenzuela et al., 2012). To complete the following series of condensation reactions, 3-ketoacyl-ACP synthase (KAS), 3-oxoacyl ACP reductase (KAR), 3-hydroxyoctanoyl-ACP dehydratase (HAD) and enoyl-ACP reductase (EAR) are involved, and finally acyl-protein thioesterase (TE) cleaves FA-ACP to release free FA. In LS-2, KAS I, KAR and HAD genes showed significant downexpression at P1 under high-dose CO2 conditions, followed by upexpression at P2, but the expression declined at P3 (Figure 6). The single-copy ENR showed prolonged up-regulation at P1 and P2, followed by significant downexpression at P3. The results demonstrated modest increases in transcripts involved in FA biosynthesis at P2 under high-dose CO2, but an overall return to a similar transcript levels under the air aeration condition at P3 which corresponds to the extended time period of lipid accumulation. Hence, lipid
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Mechanism of lipid accumulation under high-dose CO2 7
Figure 7 Gene expression changes related to carbon flow from CO2 to TAG synthesis during high-dose CO2 aeration compared with air aeration at P3. Differences in fold change are based on log2 scale and the colour scale represents differentially expressed genes. Red (log2fold change ≥1), black (log2fold change = 0.99– 0.99), Blue (log2fold change≤ 1). Dotted arrows represent undetected genes.
accumulation may be largely independent of de novo fatty acid synthesis. We speculate that supplying of substrate not the key enzymes involved in the FA biosynthesis might play a more significant role in TAG synthesis.
TAG synthesis was enhanced by high dose CO2 The free FAs generated in the chloroplast were exported to cytosol where the esterification was performed to form the acyl-CoA, a key substrate for TAG biosynthesis. The first committed step in the TAG biosynthesis is catalyzed by glycerol 3-phosphate acyltransferase (GPAT) at the sn-1 position of G-3P to produce 1-acyl-sn-glycerol-3-phosphate (LPA), subsequent LPA is converted to phosphatidic acid (PA) via 1-acyl-snglycerol-3-phosphate acyltransferase (LPAAT) at the sn-2 position. Three GPAT genes and two LPAAT genes were identified in LS-2 (Table S1), and three GPAT genes showed the same responses to high-dose CO2 aeration. Their expression was upregulated at P3, particularly GPAT-c which showed remarkable upexpression (Figures 6 and 7). Two putative LPPAT genes showed different responses to high dose CO2 aeration. One isoform was up-regulated, whereas the other one showed similar expression compared with air aeration at P3. Phosphatidic acid phosphatase (PAP) subsequently catalyzes the conversion of PA to form diacylglycerol (DAG), which was used an acyl-acceptor to form TAG via diacylglycerol acyltransferase (DGAT). DGAT has two homologs, DGAT1 (cholesterol acyltransferase family) and DGAT2 (monoacylglycerol acyltransfer-
ase family). In LS-2, one unique sequence was identified as DGAT1 and eight unique sequences were identified as DGAT2 (Table S1). DGAT genes showed the similar responses to high doses of CO2. Almost every gene was up-regulated at P2 and P3 compared with air aeration. A single-copy gene encoding PAP was also identified in LS-2, and the transcriptional analysis revealed that PAP was upexpressed at P2 and P3. Taken together, the up-regulation of the putative genes in TAG biosynthesis at P2 and P3 under high-dose CO2 presumably led to augmented TAG.
Inhibitors of GPDH and PDHC blocked lipid production of C. sorokiniana As we mentioned earlier, the elevated CO2 enhanced the carbon fixation, chloroplast glycolytic pathway, biosynthesis of acetyl-CoA, and TAG synthesis. To further validate these results, we used two specific inhibitors of GPDH and PDHC, sodium iodoacetate (NaIDO) and 3-bromopyruvic acid (3-BrPA) to target these steps. GPDH and PDHC are key genes in glycolytic pathway and biosynthesis of acetyl-CoA, and they both showed a significant up-regulated expression under highdose CO2 aeration. As expected, lipid production was reduced by 35.9% (P < 0.05) and 19.1% (P < 0.05) when 200 lM sodium iodoacetate and 1 mM 3-bromopyruvic acid were added (Figure S3). The data indicated that high-dose CO2 can indeed increase lipid production by promoting the biosynthesis of acetyl-CoA.
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Discussion Attempts to stimulate TAG generation by genetic engineering technology have long been attempted (Beer et al., 2009). For instance, the metabolic engineering has been investigated by overexpression of acc, but overexpression did not result in an increase of overall lipid biosynthesis (Courchesne et al., 2009). Recently, transcriptome analysis reveals that carbon precursors from protein and carbohydrate were shunted into FA synthesis under N deprivation, resulting in increased overall TAG production (Li et al., 2014). This regulation mechanism provided a basis for understanding TAG synthesis in microalgae and will also enable more rational genetic engineering of lipid production. However, this stress condition would also presumably limit growth of microalgal cells, which is nondesirable (Mata et al., 2010). An alternative approach would be to supply microalga with extra CO2 for scale-up lipid production as has been done previously (Reijnders, 2013; Shen, 2014). However, the molecular and cellular mechanisms underlying lipid accumulation under high doses of CO2 have not been fully elucidated. Identifying the pathways and regulatory mechanism would further guide the rational genetic engineering of microalgae for the overproduction of TAG. Ten percent CO2 aeration cultures showed up to 2.23fold increases in gClipid/Ctotal than air aeration conditions, suggesting that more carbon might flow into the lipid synthesis pathway. The pathway of carbon flow includes the generation of carbon precursor acetyl-CoA, FA biosynthesis and TAG biosynthesis (Valenzuela et al., 2012). Global gene expression analysis revealed that high doses of CO2 induced the upexpression of key genes involved in carbon fixation, carbohydrate metabolism and the TCA cycle, but the downexpression of FA biosynthesis pathway genes. Therefore, high doses of CO2 may drive more carbon flow into FA biosynthesis with the enhanced supply of carbon skeletons and reducing power. Cells maintained upexpression of key genes involved in carbon flow from CO2 to acetyl-CoA, containing the Calvin cycle, glycolysis and PHDC pathways, indicating that carbon flow increases into acetyl-CoA could be an initial trigger for lipid accumulation. Both chloroplastic and cytosolic glycolysis occurred in LS-2, but only chloroplastic glycolysis was enhanced at the prolonged lipid accumulation phases, suggesting that the increase of lipids could result of accelerated partitioning of newly photosynthetically fixed carbon through chloroplastic glycolysis but not cytosolic glycolysis. In addition, the transporter genes responsible for importing PEP and pyruvate into the chloroplast from the cytosol were up-regulated under high doses of CO2, indicating that supply of precursors from the cytosol also accelerated FA synthesis. The lipid accumulation at P2 coincided with upexpression for ACCase, KAS and KAR. However, the expression for these genes declined during P3 when lipids were being accumulated at a faster rate. These results could suggest that carbon may also be ‘pushed’ into FA via carbohydrate metabolism by high doses of CO2. When excess carbon is supplied, rapid growth of the cells may consume more nitrogen, which results in a significant increase in C/N (Reijnders, 2013; Shen, 2014). This case may mimic the N deprivation state and therefore LS-2 may exhibit a similar response mechanism. In contrast to N deprivation, genes involved in TAG biosynthesis were also upexpressed at the prolonged lipid accumulation phases under high dose CO2. Hence, except for being ‘pushed’ by carbon flow, being ‘pulled’ by a large abundance of TAG
synthesis activity may also play a significant role in enhanced fatty acid biosynthesis under high doses of CO2. With respect to biofuel production, an improved understanding of how carbon is potentially ‘pushed’ into FA biosynthesis or TAG synthesis ‘pulls’ carbon flow will promote the development of metabolic networks that can be engineered to maximize inorganic carbon flow into lipid production under high-dose CO2.
Experimental procedures Algal species and culture Chlorella sorokiniana LS-2 (saved in China General Microbiological Culture Collection Center, CGMCC 8710) was used in all experiments. C. sorokiniana LS-2 was grown at 26 °C in 7-L photobioreactors in BG-11 medium. Cells were grown under 100 lE/m2/s illumination with a photoperiod of 12 h of light followed by 12 h of dark. The photobioreactors were aerated with 10% CO2 (v/v) or with air as the control. The biomass yield was determined by measuring dry cell weight. Cells were withdrawn daily and centrifuged at 4000 g for 5 min. The precipitation was washed twice with deionized water and dried at 60 °C until constant weight. For use in mRNA sequencing, cells at exponential phase were collected (10 000 g, 5 min at 4 °C). The supernatant was discarded and the collected cells were immediately flash frozen in liquid nitrogen until further processed for RNA extraction. When quantitative PCR (qPCR) was performed, algal cells were harvested at early exponential (P1), transition from exponential to stationary phase (P2) and stationary phase (P3). The samples were stored in liquid nitrogen for total RNA extraction.
RNA extraction, library preparation and sequencing mRNA-seq libraries of C. sorokiniana were constructed according to protocols provided by Illumina. In brief, total RNAs were isolated using the TRIzol reagent (Life Technologies, Carlsbad, California) and were then treated with RNAase-free DNase I (Takara, Japan) to remove any contaminating genomic DNA. mRNA was extracted using Dynabeads oligo (dT) (Dynal; Life Technologies) and then was fragmented. Double-stranded cDNAs were then synthesized using reverse transcriptase (Superscript II; Life Technologies) and random hexamer primers. The standard Illumina protocol was followed thereafter to develop mRNA-seq libraries. Subsequently, the library preparations were sequenced on an Illumina HiSeq2000 platform and 100-bp paired-end reads were generated.
Data processing assembly and annotation The raw reads were cleaned by removing reads containing adapter, poly-N and low-quality reads. Assembly of sequences was performed with Trinity (v2012-10-05). The longest transcript of each subcomponent was defined as the ‘unigene’ for functional annotation. All the assembled unigenes of the two samples were searched against Nr (Nonredundant protein database), Nt (Nonredundant nucleotide sequences), and SwissProt, using the Blast algorithm with a E-value cut-off of 10 5 and KOG (Eukaryotic Orthologous Groups of proteins) database with a Evalue cut-off of 10 3. PFAM protein family alignments were performed using the HMMER 3.0 package. Gene ontology (GO) classification of each gene model was carried out using Blast2GOv2.5 and KEGG classification was performed using KASS and the KEGG Automatic Annotation Server, respectively. Targeting prediction was performed using TargetP 1.1 Server and SignalP 4.1 Server (Ouyang et al., 2013).
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Mechanism of lipid accumulation under high-dose CO2 9 Analysis of gene expression patterns The read counts of a gene in each sample were estimated by mapping clean reads to the Trinity transcripts assembled by RSEM (Li and Dewey, 2011). The abundance of all genes was normalized and calculated using uniquely mapped reads by the PRKM method (Mortazavi et al., 2008). Read count was normalized with the TMM method, and then differential expression analysis of the two samples was performed using DEGseq method with the threshold q-value of 2 demarcating significantly different expression levels (Wang et al., 2010). GO and KO enrichment analyses were performed based on the identified differentially expressed genes. GO-enrichment analyses were carried out using the GPseq method based on the Wallenius noncentral hypergeometric distribution with P-value of
doi: 10.1111/pbi.12398
Elevated CO2 improves lipid accumulation by increasing carbon metabolism in Chlorella sorokiniana Zhilan Sun, Yi-Feng Chen and Jianchang Du* Institute of Biotechnology, Provincial Key Laboratory of Agrobiology, Jiangsu Academy of Agricultural Sciences, Nanjing, China
Received 27 January 2015; revised 14 April 2015; accepted 16 April 2015. *Correspondence (Tel +86-025-84392767; fax +86-025-84392767; email [email protected])
Keywords: Chlorella sorokiniana, photosynthesis, carbon dioxide, transcriptomics, lipid, carbon flow.
Summary Supplying microalgae with extra CO2 is a promising means for improving lipid production. The molecular mechanisms involved in lipid accumulation under conditions of elevated CO2, however, remain to be fully elucidated. To understand how elevated CO2 improves lipid production, we performed sequencing of Chlorella sorokiniana LS-2 cellular transcripts during growth and compared transcriptional dynamics of genes involved in carbon flow from CO2 to triacylglycerol. These analyses identified the majority genes of carbohydrate metabolism and lipid biosynthesis pathways in C. sorokiniana LS-2. Under high doses of CO2, despite down-regulation of most de novo fatty acid biosynthesis genes, genes involved in carbohydrate metabolic pathways including carbon fixation, chloroplastic glycolysis, components of the pyruvate dehydrogenase complex (PDHC) and chloroplastic membrane transporters were upexpressed at the prolonged lipid accumulation phase. The data indicate that lipid production is largely independent of de novo fatty acid synthesis. Elevated CO2 might push cells to channel photosynthetic carbon precursors into fatty acid synthesis pathways, resulting in an increase of overall triacylglycerol generation. In support of this notion, genes involved in triacylglycerol biosynthesis were substantially up-regulated. Thus, elevated CO2 may influence regulatory dynamics and result in increased carbon flow to triacylglycerol, thereby providing a feasible approach to increase lipid production in microalgae.
Introduction Nutrient limitation (deprivation of nitrogen or phosphorus) is the primary method used to enhance lipid production in microalgae (Yang et al., 2013). For example, the lipid content of Chlorella vulgaris grown under nitrogen deprivation was estimated to be up to 70% (Amaro et al., 2011). Likewise, when subjected to nitrogen or phosphorus limitation, Scenedesmus sp. showed an increase in lipids as high as 30% and 53%, respectively (Xin et al., 2010). It is widely accepted that when there is insufficient nitrogen for protein synthesis, excess carbon from photosynthesis is channelled into storage molecules such as triacylglycerols (Griffiths et al., 2014). In general, this would limit cell growth while increasing lipid content (Mata et al., 2010). Therefore, a two-stage process was proposed for photosynthetic microalgae. In the first stage of this process, cells are first grown under nutrient sufficient conditions to obtain high biomass accumulation. In the second stage of this process, cells are grown under nutrient limited conditions to enhance lipid synthesis (Rodolfi et al., 2009). The mechanism of lipid accumulation under nutrient limitation may be that it provides cells excess carbon by depriving nitrogen (Li et al., 2014; Valenzuela et al., 2012). However, this stress condition would limit the growth of microalgal cells (Mata et al., 2010). An alternative strategy would be to provide excess carbon in the form of carbon dioxide (CO2), whereby growth and lipid accumulation might be achieved in a so-called one-stage process (Cheng et al., 2013). Extra CO2 supply is believed to be a more
promising approach for scale-up lipid production than nitrogen deprivation because microalgae may assimilate CO2 from the flue gas of factories, thereby upgrading this waste stream into usable products such as lipid (Reijnders, 2013; Shen, 2014). Microalgae are so far the only species known to be able to utilize extremely high levels of CO2 up to 20% (Solovchenko and KhozinGoldberg, 2013). For example, Botryococcus braunii could grow well under 20% CO2 even without any adjustment of culture pH (Ge et al., 2011). Scenedesmus sp. and Chlorella sp. were also reported to grow well when exposed to a gas mixture containing up to 20% CO2 (Westerhoff et al., 2010). However, the majority of previous studies have focused on the effects of medium composition, illumination strategies, or various photo-bioreactor setups on the growth and lipid production of microalgae; the mechanism of lipid accumulation under high doses of CO2 has not been fully elucidated (Pires et al., 2012). Valenzuela suggested that the build-up of precursors to the acetyl coenzyme A (acetyl-CoA) carboxylases may play a more significant role in triacylglycerol (TAG) synthesis rather than the actual enzyme levels of acetyl-CoA carboxylases under nitrogen deprivation conditions (Valenzuela et al., 2012). They hypothesized that carbon was being ‘pushed’ into fatty acid (FA) synthesis via elevated acetyl-CoA and NADPH levels and thus not being ‘pulled’ by a large abundance of FA synthesis activity (Valenzuela et al., 2012). Our previous work revealed high carbon–lipid conversion efficiencies in a high-dose CO2 culture system when compared with cultures grown under ambient CO2 (air) conditions. This suggests that more fixed carbon might flow into the
Please cite this article as: Sun, Z., Chen, Y.-F. and Du, J. (2015) Elevated CO2 improves lipid accumulation by increasing carbon metabolism in Chlorella sorokiniana. Plant Biotechnol. J., doi: 10.1111/pbi.12398
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2 Zhilan Sun et al. lipid synthesis pathway under high doses of CO2. Based on this, we proposed that carbon flow may also be driven by increasing the flux of CO2 to acetyl-CoA in microalgal chloroplasts in response to high doses of CO2 leading to increased lipid accumulation. To clarify how carbon was channelled into lipid synthesis pathways, we employed Illumina paired-end sequencing of C. sorokiniana cellular transcripts collected under variable growth conditions. Global transcriptome analysis revealed a detailed carbon metabolism pathway from CO2 to acetyl-CoA, which is the key precursor for FA biosynthesis in chloroplasts. Furthermore, transcriptional dynamics of acetyl-CoA producing associated genes provided evidence that enhanced carbon flow from CO2 to acetyl-CoA in the chloroplast might be the critical factor for lipid accumulation under high doses of CO2. Additionally, transcriptional dynamics analysis revealed that TAG synthesis-associated genes could be another factor involved in enhanced lipid accumulation. These findings improve our understanding of lipid production in microalgae under high doses of CO2 and will provide a new platform for future genetic engineering in microalgae for the overproduction of lipid.
carbohydrate metabolism (319), amino acid metabolism (257), cofactor and vitamin metabolism (189), and lipid metabolism (159) (Figure 3). Pathways related to translation, organismal systems, cellular processes and environmental information processing were also well represented by unigenes from C. sorokiniana LS-2. These data provided a valuable resource for investigating metabolic pathways in C. sorokiniana LS-2 cells when grown under air or CO2 aeration. Of the global metabolic pathway, our particular attention would focus on the energy and substance metabolism including photosynthesis, glycolysis, tricarboxylic acid cycle (TCA cycle), FA and TAG biosynthesis because C. sorokiniana LS-2 had the potential to accumulate lipid under high doses of CO2. Additionally, the assigned photosynthesis pathway suggested that C. sorokiniana possessed some unique features for the C4-like photosynthesis pathway. The C4 pathway is believed to improve photosynthetic carboxylation and reduce photorespiration via increasing CO2 flux to ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Therefore, the regulation of C4-like pathway response to high doses of CO2 was also investigated below.
A functional C4-like pathway exists in C. Sorokiniana
Results Sequencing, de novo assembly, and functional annotations To investigate the potential metabolic pathways involved in enhanced lipid synthesis under high doses of CO2, two sequencing libraries at the exponential phases were prepared from C. sorokiniana LS-2 and sequenced with the Illumina paired-end HiSeq platform. Growth of LS-2 under air and 10% CO2 aeration was monitored by biomass measurement (Figure S1). There were 125,162,732 clean reads generated from C. sorokiniana LS-2. Of the clean reads, 94.96% bases had a Q-value ≥20. All the raw data have been submitted to SRA database (SRP056572). De novo assembly generated 22 432 unigenes with an average length of 977 bp and N50 of 1680 bp. This Transcriptome Shotgun Assembly project has been deposited at DDBJ/EMBL/ GenBank under the Accession Number GCUV00000000. The version described in this study is the first version, GCUV01000000. Of these 22 432 unigenes, 45.52% were annotated with GO terms. They were classified into 3 functional categories: molecular function, biological process and cellular component (Figure 1). The matched unique sequences assigned to molecular function were clustered into 12 classification bins with the largest subcategory involved in ‘binding’ (68.35%) and the second largest subcategory involved in ‘catalytic activity’ (21.35%). The unique sequences were classified into 20 bins with the most abundant bin comprising transcripts involved in metabolic (53.5%) and cellular processes (20.9%). In terms of cellular components, the unique sequences were divided into 14 classifications with the most abundant being classified as ‘cell part’ (30.0%) and ‘cell’ (19.7%). The 5163 matched unique sequences were divided into 26 categories, using KOG proteins (Figure 2). The dominant category includes general functional prediction, post-translational modification, protein turnover and chaperon, and signal transduction, respectively. For further detailed understanding of their function, unique sequences were then analyzed for KO identifiers using the KEGG database. Of the 5163 matched unique sequences, 4666 of the KO annotated unigenes were capable of being assigned to KEGG biochemical pathways. The most well-represented metabolic pathways are involved in energy metabolism (329),
Rubisco is the central carboxylation enzyme for CO2 fixation during photosynthesis, but its affinity for CO2 is low (Giordano et al., 2005). To solve the problem, several microalgae have developed a C4-like CO2 concentration mechanism (CCM) to maintain photosynthetic carboxylation when cells encounter low CO2 availability (Chang et al., 2013; Haimovich-Dayan et al., 2013). In the C4-like pathway, relatively high affinity carboxylases fix inorganic carbon into C4 compounds that can be transported to the chloroplast. The C4 intermediates are then decarboxylated to deliver the inorganic carbon to the relatively low-affinity Rubisco enzyme (Worden et al., 2009). Until now, the C4-like pathway has been identified in Phaeodactylum tricornutum (Kroth et al., 2008), Thalassiosira pseudonana (Reinfelder et al., 2004), Ostreococcus tauri (Derelle et al., 2006), Myrmecia incisa and Micromonassp (Ouyang et al., 2013; Worden et al., 2009). However, no similar pathway was reported in Chlorella. According to the transcriptome analysis, our data showed that a C4-like photosynthesis might exist in C. sorokiniana LS-2 (Figure 4 and Table S1). C. sorokiniana appears to encode the enzymes phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) and pyruvate orthophosphate dikinase (PPDK, EC 2.7.9.1). Each of these enzymes is necessary for C4-metabolism. A unique sequence (comp7826_c0) encoding PEPC has been identified. The signal and transit peptide were not found in this PEPC according to subcellular localization analysis, indicating that it might function within the cytosol to produce oxaloacetic acid (OAA). Subsequently, OAA may be transferred to the mitochondria or chloroplast for further decarboxylation. On the one hand, OAA was decarboxylated to pyruvate by malate dehydrogenase (MDH) and the downstream malic enzyme (ME). Three unique sequences encoding MDHNAD+ (comp10051_c0 and comp10035_c0) and ME-NAD+ (comp7698_c0) were identified, and they possessed a predicted mitochondrial transit peptide, respectively, according to subcellular localization analysis. OAA could also be decarboxylated to phosphoenolpyruvate (PEP) by a mitochondria-localized PEPCK. Two unique sequences (comp3474_c0 and comp9353_c0) were identified to encode PEPCK, including one that had 71% amino acid identity with Auxenochlorella protothecoides [GenBank:
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Mechanism of lipid accumulation under high-dose CO2 3
Figure 1 Gene ontology classification of assembled unigenes. The 22, 432 matched unigenes were classified into 3 functional categories: molecular function, biological process and cellular component.
could be carboxylated to OAA again by a pyruvate carboxylase (PYC) in mitochondria. Subsequently, the resulting OAA could participate in the TCA cycle. On the other hand, both chloroplast-localized MDH-NADP+ and its downstream enzyme NADP+-dependent ME were also identified in C. sorokiniana LS-2. Therefore, the OAA is assumed to be reduced to pyruvate with the help of the two enzymes, which target the chloroplast. The product of the decarboxylation is CO2 which can be subsequently fixed by Rubisco. Afterwards, the pyruvate may be converted by PPDK to PEP, which is then transported by PEP/Pi translocators to the cytosol, where carboxylation is performed by PEPC. A unique sequence (comp3367_c0) encoding PPDK had been identified. The subcellular localization of PPDK is unknown due to the lack of N-terminal sequence; however, the identified PEP/Pi translocator in the chloroplast implies that there should be one chloroplastic PPDK in C. sorokiniana. In summary, the identification of key enzymes indicates that a functional C4 pathway might exist in C. sorokiniana. According to the localization of those crucial enzymes, it may have two pathways for CO2 production via decarboxylation of OAA and malate in the mitochondria and chloroplast. However, CO2 utilization by Rubisco only occurs in the chloroplast. The C4-like photosynthesis pathway has long been believed to enable the microalgae to adapt to a low CO2 level habitat. Additionally, the energetic cost of a futile cycle in mitochondria implied that the C4 metabolism may have a function under excess light energy. Those suggest that the flow of metabolites in this pathway would be affected by CO2 concentration and light intensity.
Mechanism of lipid accumulation with elevated CO2 supply Figure 2 KOG functional classification of all unigenes. A total of 5163 unigenes showed significant similarity to the sequences in KOG databases and were clustered into 26 categories.
KFM29076.1]. Moreover, this identified unique sequence revealed an OAA and an ATP-binding site (Trapani et al., 2001). Additionally, a unique sequence (comp3865_c1) encoding pyruvate carboxylase (PYC) was identified and it possessed a predicted mitochondrial transit peptide. Therefore, the generated pyruvate
To clarify the molecular mechanism of lipid accumulation under high doses of CO2, we compared patterns in global gene expression of exponentially grown cells of strain LS-2 under air and CO2 aeration via mRNA-Seq. The transcriptomes were validated by qPCR analysis of 61 selected genes, and the coefficient of determination (R2) between qPCR-based and mRNA-Seq-based transcript abundance was 0.802 (Figure S2). Most of the selected genes showed a positive correlation between mRNA-Seq and qPCR (Figure S2). The significant DEGs
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Figure 3 KEGG classification of assembled unigenes. The 4666 KO annotated unigenes were assigned to 5 KEGG biochemical pathways: metabolism, genetic information processing, organism system, cellular processes and environmental information processing.
Figure 4 Model of the C4-like pathway in C. sorokiniana LS-2 based on the transcriptome. The dashed line indicates the route that was not detected in this transcriptome.
(different expression genes) were then identified with the applied criteria [q-value 1] (Figure 5). 1837 genes (8.2% of total) were found to be significantly upregulated, whereas 990 genes (4.4% of total) were downregulated in response to high CO2 aeration (Data S1). All DEGs were mapped to terms in the KEGG database. Because of the particular interest of this study in lipid synthesis under high-dose CO2 utilization, the genes involved in carbon fixation, carbohydrate metabolism and lipid synthesis were further studied (Table S1). A total of 87 differentially expressed genes related with carbon flow to lipid accumulation were identified. Among them, 51 genes (58.6%) were found to be significantly up-regulated, whereas 36 genes (37.2%) were down-regulated in response to high dose CO2 aeration. Fortynine up-regulated genes were particularly enriched in carbon fixation, carbohydrate metabolism, accounting for almost all of the deferentially expressed genes. However, down-regulated genes were mainly enriched in FA and TAG biosynthesis pathway. Of 13 differentially expressed genes in the two pathways, 11 genes were down-regulated. In particular acetyl-CoA carboxylase,
the first key enzyme involved in FA biosynthesis was downexpressed by 3.2- to 17.9-fold. TCA cycle genes were upexpressed (from 2.3- to 95.8-fold) under high-dose CO2 aeration conditions (Table S1). Levels of transcripts encoding the two potentially rate-limiting enzymes, citrate synthase and isocitrate dehydrogenase, in the TCA cycle increased 11.5- and 11.9-fold, respectively. To our knowledge, the glycolysis generated acetyl-CoA can be broken down into CO2 via the TCA cycle to produce NADH that fuels ATP synthesis by oxidative phosphorylation in the mitochondria (Sweetlove et al., 2010). Moreover, the intermediates of the TCA cycle (e.g. 2-oxoglutarate, succinyl CoA, fumarate and oxaloacetate) are linked to amino acid metabolism (Li et al., 2014). Thus, increases in carbon flow through the TCA cycle may supply the assimilatory power for carbon fixation and lipid biosynthesis, and also increase the carbon flow into these TCA cycle intermediates for amino acid biosynthesis. Besides, malate, an intermediate of the TCA cycle, can be converted to pyruvate by a NAD+-malic enzyme (ME). Pyruvate, the key precursor for acetyl-CoA can subsequently be transported to chloroplast for FA biosynthesis.
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Mechanism of lipid accumulation under high-dose CO2 5 pyruvic acid (decarboxylate of MDH and ME) is an important precursor of the TCA cycle. Therefore, we speculate that the C4-like pathway might be more likely to function in replenishing TCA cycle intermediates for energy production as opposed to functioning to redirect CO2 into carbon fixation in the chloroplast. Additionally, the transcriptional dynamics of genes involved in the Calvin cycle were also examined by qPCR. They were upexpressed at P2 and P3 in the CO2 treatment with the exception of FBP, which exhibited similar expression in the CO2 and air treatments (Figures 6 and 7).
Elevated CO2 enhances the chloroplast glycolytic pathway
Figure 5 Comparison of expression patterns of differentially expressed unigenes identified between high-dose CO2 and air aeration. The red dots represent DEGs, and the blue dots represent non-DEGs.
Therefore, the up-regulation of the TCA reactions, coupled with the increased expression of genes involved in carbon fixation, may suggest that enhanced availability of carbon skeletons and reducing power for FA and TAG synthesis can be influenced by the availability of CO2. Taken together, these results could be consistent with the previous work that carbon is being ‘pushed’ into FA synthesis via elevated acetyl-CoA and NADPH levels and thus not being ‘pulled’ by a large abundance of FA synthesis activity (Valenzuela et al., 2012). Regulation of the genes involved in carbon flow from CO2 to lipid accumulation will be discussed in detail below.
Carbon precursor supply for FA synthesis was enhanced by high-dose CO2 Carbon fixation was enhanced by high-dose CO2 Transcriptomic analysis indicated that CO2 assimilation was slightly enhanced under the high-dose CO2 condition. Most genes in the C4-like pathway and Calvin cycle were up-regulated during exponential phase growth (Table S1). To further examine the transcriptional dynamics of those genes, qPCR was performed at the P1, P2 and P3 points of the growth cycle (Figure S1 and Table S2). According to NormFinder analysis, the stability value of candidate reference gene Actin, 18S rRNA, and Tubulin was 0.013, 0.037 and 0.041, respectively, indicating that they all exhibited the relative stability in expression level. To facilitate our further analysis, Actin was used as a reference gene. Under highdose CO2 aeration, two key enzymes PEPC and PPDK in C4-like pathway were significantly upexpressed at P1 and P2, however, both of them returned to the basal level at P3 (Figures 6 and 7). The data suggest that the C4-like pathway was active during cell growth phase (P1 and P2), but played a less important role during prolonged lipid accumulation (P3). Interestingly, mitochondrial MDH and ME were upexpressed at P2 and P3; however, no similar regulation was observed in chloroplast MDH and ME. Both of these enzymes are involved in CO2 formation and consider that
Glyceraldehyde-3-phosphate (G3P), an intermediate product of the Calvin cycle, is converted to pyruvate via glycolysis. The pyruvate is then converted to acetyl-CoA, the precursor of de novo FA biosynthesis. In Nannochloropsis, glycolytic pathways operate in both the plastid and the cytosol, which produces pyruvate for FA biosynthesis while generating the high-energy compounds ATP and NADH (Li et al., 2014). All the components of the glycolytic pathway were identified in C. sorokiniana, and subcellular localization analysis of these components revealed that glycolysis is likely to take place both in the cytosol and chloroplast. The transcriptional dynamics of genes involved in glycolysis were examined by qPCR (Table S2, Figures 6 and 7). At P2, most of the glycolytic genes in the cytosol and chloroplast were upexpressed under the CO2 condition, suggesting a more active metabolic activity at P2 under high doses of CO2. As the cells went into stationary phase (P3), genes located in the chloroplast remained up-regulated under high-dose CO2. In contrast, PGK and one PGAM in cytosol were significantly down-regulated. This suggests that enhanced FA synthesis under high CO2 may be due to up-regulation of glycolysis in chloroplasts, which could supply more carbon precursors, ATP and reducing equivalents for FA synthesis when CO2 is not limiting. Additionally, genes encoding transporters that direct carbon precursors to FA biosynthesis in chloroplasts were also identified. Bile acid:Na+ symporter (BASS) mediates sodium-coupled pyruvate import into plastids (Furumoto et al., 2011). Three unique sequences (Table S1) encoding a putative BASS were identified in LS-2. In this pathway, the balance of Na+ influx is maintained by a sodium:proton antiporter (NHD) (Li et al., 2014). Thirteen NHD isoforms were identified in LS-2. Transcriptional dynamics analyses revealed that selected BASS and NHD were up-regulated under high dose CO2 at P2 and P3. A putative phosphate/ phosphoenol pyruvate translocator (PPT), which imports cytosolic PEP into the plastid stroma in exchange for inorganic phosphate (Li et al., 2014), was identified and its transcript level also increased at P2 and P3. Therefore, the evidence collectively suggests that enhanced FA synthesis under high-dose CO2 was correlated with the up-regulation of transporter genes responsible for importing the intermediates (e.g. PEP and pyruvate) from the cytosol into the chloroplast.
High CO2 enhances biosynthesis of acetyl-CoA in chloroplast Similar to plant, in microalgae, palmitic acid (16:0) is believed to be synthesized de novo in the chloroplast from the precursor acetyl-CoA. There are two possible processes for supplying acetylCoA to the chloroplast. One mechanism is the direct catalysis of pyruvate by the plastid-localized pyruvate dehydrogenase complex (PDHC), which bridges the glycolysis and lipid biosynthesis pathways in the plastid. The other mechanism is an indirect regeneration from the imported cytosolic acetate (Oliver et al.,
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Figure 6 Transcriptional dynamics of individual genes related to the carbon flow from CO2 to TAG biosynthesis in response to high dose CO2 aeration. Relative fold differences were calculated based on the MCt method using the Actin amplification product as an internal standard.
2009). PDHC consists of three subunits: E1, E2 and E3. The plastidial PDHC genes were identified in LS-2 (Table S1), and transcriptional dynamics of E2 revealed that PDHC was significantly up-regulated at P2 and P3 (Table S2, Figures 6 and 7). Considering that lipid started to accumulate at P2, the remarkable increase of acetyl-CoA in the chloroplast at P2 and P3 might be the key factor that results in the ‘push’ of carbon into FA biosynthesis pathways by high-dose CO2. On the other hand, pyruvate can be converted to acetate by a pyruvate decarboxylase (PDC) and an aldehyde dehydrogenase (ALDH), which was called the ‘PDHC bypass’ pathway (Li et al., 2014), and then, the acetate can be transported to chloroplast where acetyl transfer was performed by acetyl-CoA synthetase. Two copies of PDC genes and three copies of ALDH genes located in the cytosol were found in LS-2. The transcriptional dynamics revealed that most of the selected genes were downexpressed in the presence of high dose CO2 (Figure 6 and 7), suggesting that the ‘PDHC bypass’ maybe not the major pathway of acetyl-CoA formation. Moreover, these data suggest that the chloroplast is the major site for acetyl-CoA formation in FA biosynthesis and not the cytosol.
Lipid accumulation is largely independent of de novo fatty acid synthesis The de novo synthesis of FAs begins with acetyl-CoA via the initial catalysis by acetyl-CoA carboxylase (ACCase). malonylCoA:acyl protein malonyltransferase (MCMT) subsequently transfer the malonyl moiety to an acyl-carrier protein (ACP)
to generate malonyl-ACP, which then enters into a series of carbon chain condensation reactions to form 16- or 18-carbon FAs. ACCase consists of four subunits encoded by accA, accB, accC and accD. Seven putative ACCase genes, two ACP genes and one copy of MCMT were identified in LS-2 (Table S1). Three selected ACCase genes as well as ACP and MCMT genes were down-regulated at P1, and subsequently, were upexpressed at P2, with expression only again declining at P3. None of these genes were upexpressed at the transcriptional level during prolonged lipid accumulation (P3). Similar results were recently reported in Phaeodactylum tricornutum, in which only modest changes in gene expression of FA metabolism genes were observed (Valenzuela et al., 2012). To complete the following series of condensation reactions, 3-ketoacyl-ACP synthase (KAS), 3-oxoacyl ACP reductase (KAR), 3-hydroxyoctanoyl-ACP dehydratase (HAD) and enoyl-ACP reductase (EAR) are involved, and finally acyl-protein thioesterase (TE) cleaves FA-ACP to release free FA. In LS-2, KAS I, KAR and HAD genes showed significant downexpression at P1 under high-dose CO2 conditions, followed by upexpression at P2, but the expression declined at P3 (Figure 6). The single-copy ENR showed prolonged up-regulation at P1 and P2, followed by significant downexpression at P3. The results demonstrated modest increases in transcripts involved in FA biosynthesis at P2 under high-dose CO2, but an overall return to a similar transcript levels under the air aeration condition at P3 which corresponds to the extended time period of lipid accumulation. Hence, lipid
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Mechanism of lipid accumulation under high-dose CO2 7
Figure 7 Gene expression changes related to carbon flow from CO2 to TAG synthesis during high-dose CO2 aeration compared with air aeration at P3. Differences in fold change are based on log2 scale and the colour scale represents differentially expressed genes. Red (log2fold change ≥1), black (log2fold change = 0.99– 0.99), Blue (log2fold change≤ 1). Dotted arrows represent undetected genes.
accumulation may be largely independent of de novo fatty acid synthesis. We speculate that supplying of substrate not the key enzymes involved in the FA biosynthesis might play a more significant role in TAG synthesis.
TAG synthesis was enhanced by high dose CO2 The free FAs generated in the chloroplast were exported to cytosol where the esterification was performed to form the acyl-CoA, a key substrate for TAG biosynthesis. The first committed step in the TAG biosynthesis is catalyzed by glycerol 3-phosphate acyltransferase (GPAT) at the sn-1 position of G-3P to produce 1-acyl-sn-glycerol-3-phosphate (LPA), subsequent LPA is converted to phosphatidic acid (PA) via 1-acyl-snglycerol-3-phosphate acyltransferase (LPAAT) at the sn-2 position. Three GPAT genes and two LPAAT genes were identified in LS-2 (Table S1), and three GPAT genes showed the same responses to high-dose CO2 aeration. Their expression was upregulated at P3, particularly GPAT-c which showed remarkable upexpression (Figures 6 and 7). Two putative LPPAT genes showed different responses to high dose CO2 aeration. One isoform was up-regulated, whereas the other one showed similar expression compared with air aeration at P3. Phosphatidic acid phosphatase (PAP) subsequently catalyzes the conversion of PA to form diacylglycerol (DAG), which was used an acyl-acceptor to form TAG via diacylglycerol acyltransferase (DGAT). DGAT has two homologs, DGAT1 (cholesterol acyltransferase family) and DGAT2 (monoacylglycerol acyltransfer-
ase family). In LS-2, one unique sequence was identified as DGAT1 and eight unique sequences were identified as DGAT2 (Table S1). DGAT genes showed the similar responses to high doses of CO2. Almost every gene was up-regulated at P2 and P3 compared with air aeration. A single-copy gene encoding PAP was also identified in LS-2, and the transcriptional analysis revealed that PAP was upexpressed at P2 and P3. Taken together, the up-regulation of the putative genes in TAG biosynthesis at P2 and P3 under high-dose CO2 presumably led to augmented TAG.
Inhibitors of GPDH and PDHC blocked lipid production of C. sorokiniana As we mentioned earlier, the elevated CO2 enhanced the carbon fixation, chloroplast glycolytic pathway, biosynthesis of acetyl-CoA, and TAG synthesis. To further validate these results, we used two specific inhibitors of GPDH and PDHC, sodium iodoacetate (NaIDO) and 3-bromopyruvic acid (3-BrPA) to target these steps. GPDH and PDHC are key genes in glycolytic pathway and biosynthesis of acetyl-CoA, and they both showed a significant up-regulated expression under highdose CO2 aeration. As expected, lipid production was reduced by 35.9% (P < 0.05) and 19.1% (P < 0.05) when 200 lM sodium iodoacetate and 1 mM 3-bromopyruvic acid were added (Figure S3). The data indicated that high-dose CO2 can indeed increase lipid production by promoting the biosynthesis of acetyl-CoA.
ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–10
8 Zhilan Sun et al.
Discussion Attempts to stimulate TAG generation by genetic engineering technology have long been attempted (Beer et al., 2009). For instance, the metabolic engineering has been investigated by overexpression of acc, but overexpression did not result in an increase of overall lipid biosynthesis (Courchesne et al., 2009). Recently, transcriptome analysis reveals that carbon precursors from protein and carbohydrate were shunted into FA synthesis under N deprivation, resulting in increased overall TAG production (Li et al., 2014). This regulation mechanism provided a basis for understanding TAG synthesis in microalgae and will also enable more rational genetic engineering of lipid production. However, this stress condition would also presumably limit growth of microalgal cells, which is nondesirable (Mata et al., 2010). An alternative approach would be to supply microalga with extra CO2 for scale-up lipid production as has been done previously (Reijnders, 2013; Shen, 2014). However, the molecular and cellular mechanisms underlying lipid accumulation under high doses of CO2 have not been fully elucidated. Identifying the pathways and regulatory mechanism would further guide the rational genetic engineering of microalgae for the overproduction of TAG. Ten percent CO2 aeration cultures showed up to 2.23fold increases in gClipid/Ctotal than air aeration conditions, suggesting that more carbon might flow into the lipid synthesis pathway. The pathway of carbon flow includes the generation of carbon precursor acetyl-CoA, FA biosynthesis and TAG biosynthesis (Valenzuela et al., 2012). Global gene expression analysis revealed that high doses of CO2 induced the upexpression of key genes involved in carbon fixation, carbohydrate metabolism and the TCA cycle, but the downexpression of FA biosynthesis pathway genes. Therefore, high doses of CO2 may drive more carbon flow into FA biosynthesis with the enhanced supply of carbon skeletons and reducing power. Cells maintained upexpression of key genes involved in carbon flow from CO2 to acetyl-CoA, containing the Calvin cycle, glycolysis and PHDC pathways, indicating that carbon flow increases into acetyl-CoA could be an initial trigger for lipid accumulation. Both chloroplastic and cytosolic glycolysis occurred in LS-2, but only chloroplastic glycolysis was enhanced at the prolonged lipid accumulation phases, suggesting that the increase of lipids could result of accelerated partitioning of newly photosynthetically fixed carbon through chloroplastic glycolysis but not cytosolic glycolysis. In addition, the transporter genes responsible for importing PEP and pyruvate into the chloroplast from the cytosol were up-regulated under high doses of CO2, indicating that supply of precursors from the cytosol also accelerated FA synthesis. The lipid accumulation at P2 coincided with upexpression for ACCase, KAS and KAR. However, the expression for these genes declined during P3 when lipids were being accumulated at a faster rate. These results could suggest that carbon may also be ‘pushed’ into FA via carbohydrate metabolism by high doses of CO2. When excess carbon is supplied, rapid growth of the cells may consume more nitrogen, which results in a significant increase in C/N (Reijnders, 2013; Shen, 2014). This case may mimic the N deprivation state and therefore LS-2 may exhibit a similar response mechanism. In contrast to N deprivation, genes involved in TAG biosynthesis were also upexpressed at the prolonged lipid accumulation phases under high dose CO2. Hence, except for being ‘pushed’ by carbon flow, being ‘pulled’ by a large abundance of TAG
synthesis activity may also play a significant role in enhanced fatty acid biosynthesis under high doses of CO2. With respect to biofuel production, an improved understanding of how carbon is potentially ‘pushed’ into FA biosynthesis or TAG synthesis ‘pulls’ carbon flow will promote the development of metabolic networks that can be engineered to maximize inorganic carbon flow into lipid production under high-dose CO2.
Experimental procedures Algal species and culture Chlorella sorokiniana LS-2 (saved in China General Microbiological Culture Collection Center, CGMCC 8710) was used in all experiments. C. sorokiniana LS-2 was grown at 26 °C in 7-L photobioreactors in BG-11 medium. Cells were grown under 100 lE/m2/s illumination with a photoperiod of 12 h of light followed by 12 h of dark. The photobioreactors were aerated with 10% CO2 (v/v) or with air as the control. The biomass yield was determined by measuring dry cell weight. Cells were withdrawn daily and centrifuged at 4000 g for 5 min. The precipitation was washed twice with deionized water and dried at 60 °C until constant weight. For use in mRNA sequencing, cells at exponential phase were collected (10 000 g, 5 min at 4 °C). The supernatant was discarded and the collected cells were immediately flash frozen in liquid nitrogen until further processed for RNA extraction. When quantitative PCR (qPCR) was performed, algal cells were harvested at early exponential (P1), transition from exponential to stationary phase (P2) and stationary phase (P3). The samples were stored in liquid nitrogen for total RNA extraction.
RNA extraction, library preparation and sequencing mRNA-seq libraries of C. sorokiniana were constructed according to protocols provided by Illumina. In brief, total RNAs were isolated using the TRIzol reagent (Life Technologies, Carlsbad, California) and were then treated with RNAase-free DNase I (Takara, Japan) to remove any contaminating genomic DNA. mRNA was extracted using Dynabeads oligo (dT) (Dynal; Life Technologies) and then was fragmented. Double-stranded cDNAs were then synthesized using reverse transcriptase (Superscript II; Life Technologies) and random hexamer primers. The standard Illumina protocol was followed thereafter to develop mRNA-seq libraries. Subsequently, the library preparations were sequenced on an Illumina HiSeq2000 platform and 100-bp paired-end reads were generated.
Data processing assembly and annotation The raw reads were cleaned by removing reads containing adapter, poly-N and low-quality reads. Assembly of sequences was performed with Trinity (v2012-10-05). The longest transcript of each subcomponent was defined as the ‘unigene’ for functional annotation. All the assembled unigenes of the two samples were searched against Nr (Nonredundant protein database), Nt (Nonredundant nucleotide sequences), and SwissProt, using the Blast algorithm with a E-value cut-off of 10 5 and KOG (Eukaryotic Orthologous Groups of proteins) database with a Evalue cut-off of 10 3. PFAM protein family alignments were performed using the HMMER 3.0 package. Gene ontology (GO) classification of each gene model was carried out using Blast2GOv2.5 and KEGG classification was performed using KASS and the KEGG Automatic Annotation Server, respectively. Targeting prediction was performed using TargetP 1.1 Server and SignalP 4.1 Server (Ouyang et al., 2013).
ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 1–10
Mechanism of lipid accumulation under high-dose CO2 9 Analysis of gene expression patterns The read counts of a gene in each sample were estimated by mapping clean reads to the Trinity transcripts assembled by RSEM (Li and Dewey, 2011). The abundance of all genes was normalized and calculated using uniquely mapped reads by the PRKM method (Mortazavi et al., 2008). Read count was normalized with the TMM method, and then differential expression analysis of the two samples was performed using DEGseq method with the threshold q-value of 2 demarcating significantly different expression levels (Wang et al., 2010). GO and KO enrichment analyses were performed based on the identified differentially expressed genes. GO-enrichment analyses were carried out using the GPseq method based on the Wallenius noncentral hypergeometric distribution with P-value of
