Biotechnol Lett DOI 10.1007/s10529-014-1593-3

ORIGINAL RESEARCH PAPER

Expression and knockdown of the PEPC1 gene affect carbon flux in the biosynthesis of triacylglycerols by the green alga Chlamydomonas reinhardtii Xiaodong Deng • Jiajia Cai Yajun Li • Xiaowen Fei

•

Received: 23 April 2014 / Accepted: 12 June 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract The regulation of lipid biosynthesis is important in photosynthetic eukaryotic cells. This regulation is facilitated by the direct synthesis of fatty acids and triacylglycerol (TAG), and by other controls of the main carbon metabolic pathway. In this study, knockdown of the mRNA expression of the Chlamydomonas phosphoenolpyruvate carboxylase isoform 1 (CrPEPC1) gene by RNA interference increased TAG level by 20 % but decreased PEPC activities in the corresponding transgenic algae by 39–50 %. The decrease in CrPEPC1 expression increased the expression of TAG biosynthesis-related genes, such as acylCoA:diacylglycerol acyltransferase and phosphatidate phosphatase. Conversely, CrPEPC1 over-expression decreased TAG level by 37 % and increased PEPC activities by 157–184 %. These observations suggest that the lipid content of algal cells can be controlled by regulating the CrPEPC1 gene.

Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1593-3) contains supplementary material, which is available to authorized users. X. Deng  J. Cai  Y. Li Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Science, Haikou 571101, China X. Fei (&) School of Science, Hainan Medical College, Haikou 571101, China e-mail: [email protected]

Keywords Chlamydomonas reinhardtii  Lipid biosynthesis  Phosphoenolpyruvate carboxylase  RNAi interference  Triacylglycerol biosynthesis

Introduction The gradual depletion of fossil fuel resources has emphasized the importance of energy and environment. Microalgae biodiesel, a crucial part of renewable biomass energy that uses solar energy to fix CO2 into biomass, is a promising alternative to fossil fuels. However, little is known about lipid metabolism in eukaryotic unicellular photosynthetic microalgae. The intensification of microalgae-derived biodiesel research at the global scale has motivated researchers to investigate the mechanisms underlying the formation of cultures with high lipid production and high cell density. Miller et al. (2010) studied the biological processes in Chlamydomonas under N-deprived conditions. They found that the genes involved in protein biosynthesis, tricarboxylic (TCA) cycle, and photosynthesis were down-regulated while those involved in triacylglycerol (TAG) biosynthesis were up-regulated. These TAG biosynthesis-related genes include phosphatidate phosphatase and acyl-CoA:diacylglycerol acyltransferase (DGAT). N deprivation also greatly influences metabolism; acetate is directly funneled into fatty acid biosynthesis and additional fatty acids are produced by membrane remodeling. These

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findings indicate that the breakdown of intracellular stores under N-deprived conditions is an efficient source of carbon for N reassimilation. Although photosynthetic carbon fixation is decreased under N-deprived conditions, some genes for glycolysis are up-regulated. This phenomenon indicates that parts of the carbon from starch are reutilized as carbon skeleton for fatty acid biosynthesis. Phosphoenolpyruvate carboxylase (PEPC, EC4.1. 1.31) is a cytoplasmic enzyme widely distributed in vascular plants, archaea, bacteria, and unicellular green algae (Ettema et al. 2004; Guy et al. 1989). This enzyme catalyzes the formation of oxaloacetate (OAA) from phosphoenolpyruvate (PEP) that then enters the TCA cycle to provide the substrate and energy for protein synthesis. Acetyl-CoA carboxylase (ACCase) catalyzes the formation of malonyl-CoA from acetyl coenzyme A and then enters the fatty acid synthesis pathway. Hence, the carbon flux from glycolysis synthesizes proteins or fatty acids depending on the relative activity of PEPC and ACCase. Two classes of PEPC complex, PEPC1 and PEPC2, have been identified in green microalgae (O’Leary et al. 2011). The former is a minor homotetramer composed of the p109 subunit; the latter is a highly abundant, high-mass, heteromer composed of the p109 and p131 subunits. The p109 and p131 subunits are coded by the CrPEPC1 and CrPEPC2 genes, respectively (Moellering et al. 2007; Mamedov et al. 2005, 2010). Our group has found that the mRNA abundance of Chlamydomonas PEPC2 negatively correlates with cellular lipid accumulation (Deng et al. 2011). However, whether or not the expression and regulation of the PEPC1 gene is related to cellular lipid accumulation remains unknown. CrPEPC1 was silenced by RNA interference (RNAi) and overexpressed in Chlamydomonas to ascertain whether or not the expression of CrPEPC1 affects cellular carbon flux and lipid accumulation. The results of this study support the existing evidence that lipid accumulation is related to carbon flux distribution.

Materials and methods Bioinformatics The information on the Chlamydomonas PEPC1 gene (g16646), the subunit of p109, was obtained from the

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Phytozome V9.1 Chlamydomonas database (http:// www.phytozome.net). Subcellular localization of proteins was predicted by Euk-mPLoc 2.0. The sequence alignment and phylogenetic tree of CrPEPC1 were created by ClustalW (http://www. genome.jp/tools/clustalw/). Active consensus sites were identified based on the Sanger Pfam database (http://pfam.sanger.ac.uk/search). Algal strain, cultivation conditions, and biomass assay Chlamydomonas reinhardtii CC425 (mt) was purchased from the Chlamydomonas Genetics Center. Cells grown on Tris/acetate/phosphate (TAP) agar plates were inoculated in Erlenmeyer flasks containing 50 ml HSM and N-deficient HSM (HSM-N) media. The HSM medium is composed of NH4Cl (0.5 g l-1), MgSO47H2O (0.02 g l-1), CaCl22H2O (0.01 g l-1), K2HPO4 (1.44 g l-1), KH2PO4 (0.72 g l-1), sodium acetate (2 g l-1), H3BO3 (0.001 g l-1), MnCl24H2O (0.005 g l-1), ZnSO47H2O (0.022 g l-1), FeSO4 7H2O (0.005 g l-1), CoCl26H2O (0.002 g l-1), Na2 Mo7O244H2O (0.002 g l-1), and sodium EDTA (0.05 g l-1). HSM-N medium contains the same components except NaCl was replaced with NH4Cl. All cultures were maintained at 25 °C and 150 lmol m-2s-1 with shaking at 230 rpm. Biomass (g l-1) was determined by measuring the OD490 value see Deng et al. (2011) and then calculated to the cell dry weight (see Supplementary Fig. 1). Lipid content analysis The Nile Red fluorescence method and GC/MS were used to determine lipid and TAG levels. Cells were directly stained with Nile Red for 10 min, and then fluorescence was measured at 470 and 570 nm, respectively. The fluorescence value was calculated using the equation: FD (470/570) = (A2–A1) (Supplementary Fig. 2). Total lipid extraction was carried out according to Liu et al. (2013). Mid-growth cells were collected by centrifugation and resuspended in lipid extraction solvent (methanol/chloroform/methanoic acid, 2:1:0.1, by volume), and then added an extraction buffer (1 M KCl and 0.2 M H3PO4), and mixed vigorously. Lipids were obtained by centrifugation at 14,0009g for 3 min. TAG was separated using Si60

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silica TLC plates. These plates were held at 120 °C for 2.5 h and cooled at room temperature. After samples were added under N2 flow, TAGs were observed on TLC plates by I2 staining (Supplementary Fig. 3) (Liu et al. 2013). Fatty acid methyl esters derived from TAG were analyzed by GC/MS (La Russa et al. 2012). For the microscopic assay, images were acquired using a fluorescence microscope after Nile Red staining. RNA extraction Total RNA was prepared as Li et al. (2012) with modifications. Cells from 10 ml cultivated algae were collected by centrifugation at 10,0009g for 1 min. After a series of phenol/chloroform extractions, nucleic acids were precipitated with 2 vol. ethanol and then washed with 75 % (v/v) ethanol. Finally, the air-dried pellet was dissolved in 40 ll RNase-free water. Cloning of the CrPEPC1 gene First-strand cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen, USA). A fragment of the CrPEPC1 gene was amplified by PCR using primers CrPEPC1L: 50 -CAGCTGTCTGCTA CCAGCGGC-30 and CrPEPC1R: 50 -TAAGATCGT TTTGCTCGCGT-30 . After purification by column DNA gel extraction kit (BBI, Canada), the DNA was inserted into the vector pMD18-T (TaKaRa, Japan). The resulting plasmid was designated as pMD18TCrPEPC1. The sequence of the cloned CrPEPC1 gene was verified by sequence analysis. Construction of the RNAi vector against the CrPEPC1 gene The gene sequences of CrPEPC1 and g11831 are almost identical, and CrPEPC1 only has an additional 59 amino acid sequence between sites 380 and 439 than g11831. Hence, we selected the DNA sequence in this part to construct the RNAi vector against the CrPEPC1 gene. A fragment of the C. reinhardtii 18S gene was amplified with primers 50 -CGAACTTCTG CGAAAGCAT-30 and 50 -TCAGCCTTGCGACCA TACT-30 and then inserted into pMD18-T to produce pMD18T-18S. The fragment of CrPEPC1 and its reverse complementary sequences were amplified by

PCR using pMD18T-CrPEPC1 as a template. The primers for this reaction were CrPEPC1RNAiL: 50 GTGCTGCACCAGTGCCTCA-30 and CrPEPC1R NAiR: 50 -CTGTCGCAGCAGGTCCAGCAG-30 . The PCR fragment was digested with KpnI/BamHI and HindIII/SalI, and then inserted into the corresponding cloning sites of pMD18T-18S to yield pMD18CrPEPC1F-18S-CrPEPC1R, which contained an inverted repeat sequence of CrPEPC1 (CrPEPC1 IR). pMD18-CrPEPC1F-18S-CrPEPC1R was doubledigested with KpnI and HindIII to obtain CrPEPC1 IR. Finally, CrPEPC1 IR was inserted as a blunt-end fragment into EcoRI-digested pMaa7/XIR to yield pMaa7IR/CrPEPC1 IR. Construction of the over-expression vector of CrPEPC1 for Chlamydomonas To construct the over-expression vector of CrPEPC1, the coding sequence of CrPEPC1 was amplified by PCR using pMD18T-CrPEPC1 as a template. The primers used for this reaction were 50 -TAGTAGA TCTGATGGACGCGGTGACCAC-30 and 50 -TATA ACTAGTTCACCCCGTGTTCTGCATGCC-30 . The fragment was digested with NcoI/SpeI and inserted into similarly digested pCAMBIA1302 to yield pCAMPEPC1, which allows CrPEPC1 over-expression. Chlamydomonas transformation Chlamydomonas reinhardtii was transformed as previously described by Kindle (1990). Cells were grown in TAP medium to 2 9 106 cells ml-1. The cells were collected by centrifugation, and then resuspended in TAP medium. Plasmid DNA was introduced into the cells by the glass bead procedure. In each case, 2 lg plasmid DNA was included in a mixture containing 400 ll cells, 100 ll 20 % (v/v) polyethylene glycol, and 300 mg sterile glass beads. The reaction was mixed for 15 s on a benchtop vortex. To allow induction of RNAi or gene expression, the cells were allowed to recover for 1 day before plating onto selective media. RNAi transformants were selected on TAP medium containing 1.5 mM Ltryptophan, 5 lg paromomycin ml-1 and 5 lM 5-FI. pCAMPEPC1 transformants were selected on TAP medium containing 50 lg hygromycin ml-1. The plates were incubated under dim light (*50 lmol m-2 s-1 photosynthetically active radiation). Isolated transgenic strains were kept under constant selective pressure.

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CrPEPC1 expression in E. coli BL21 To express CrPEPC1 in E. coli BL21, the coding region was amplified from pMD18T to CrPEPC1 using the primers GEXPEPC1-F: 50 -TTTAGGATC CATGCAGCTGTCTGCTACCAGCGGC-30 and GEXPEPC1-R: 50 -AAATGTCGACTCACCCCGTG TTCTGCATGCC-30 . The amplified fragments were digested with BamHI/SalI and inserted into similar digested pGEX-6p-1 to yield pGEXPEPC1. The transformation of E. coli BL21 and subsequent foreign protein detection by SDS-PAGE and purification of the GST-fusion protein were conducted by standard protocol. Quantitative real-time PCR Real-time PCR analysis was performed as previously described by Deng et al. (2011). RNA was extracted using TRIzol. cDNA was synthesized with an Invitrogen SuperScript cDNA synthesis kit using 100 ng RNA and random primers. Real-time PCR was performed on a BioRad iCycler iQ Real-Time PCR Detection System using SYBR Green as the fluorescent dye. 18S rRNA was used as a control with the primers 18S rRNAF (50 -TCAACTTTCGATGGTAGGATAGTG-30 ) and 18S rRNAR (50 -CCGTGTCAGGATTGG GTAATTT-30 ). Gene-specific primers (Supplementary Table 1) were used to evaluate the quantity of the target cDNA. The amplification rate of each transcript (Ct) was calculated by the PCR baseline-subtraction method performed in the iCycler software at a constant fluorescence level. Cts were determined over three repeats. Relative fold differences were calculated based on the relative quantification analytical method (2-DDCt) (Livak and Schmittgen 2001). Detection of PEPC activities Transgenic algal samples, cultivated in the midgrowth phase, were collected by centrifuging. Cell pellets were washed with phosphate buffer, sonicated for 40 s, then centrifuged at 3,0009g for 5 min. The supernatant was collected and dialyzed with extraction buffer (0.2 M Tris–HCl, pH 8.2, 10 mM D-isoascorbic acid, 0.1 % Triton X-100) overnight. PEPC activity was measured as described by Hirai and Ueno (1977) with modifications. The enzyme extraction was mixed

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with 40 mM reaction buffer (2 mM MgCl2, 10 mM KHCO3, 2 mM PEP, 0.5 mM GSH, 0.15 mM NADH, pH 8.5). The absorbance of the samples was measured at 340 nm. One unit of enzyme was defined as the amount of enzyme required to cause an absorbance change of 0.01 per min.

Results Cloning of the CrPEPC1 gene and bioinformatics analysis The p109 subunit gene (CrPEPC1) sequence reported by Mamedov et al. (2005) (NCBI: AY517644) was used for entry in BlastP in the Phytozome V9.1 C. reinhardtii database. The g16646, g11831, and Cre03.g171950 genes with identities of 100, 93.5, and 47.6 % were identified, respectively. The CrPEPC1 gene (g16646) is located on chromosome 16 between sites 6,387,917 and 6,402,662, whereas the g11831 gene is located on chromosome11 between sites 2591855 and 2606468. The CrPEPC1 gene cloned in this study is 100 % identical to the g16646 gene, which is highly similar to the g11831 gene; that is, only 59 amino acids from sites 380 to 439 are deleted. The Cre03.g171950 gene is the p131 subunit (CrPEPC2) gene (Mamedov et al. 2005). Using the BLAST program and the Chlamydomonas PEPC1 gene as entries, we obtained the CrPEPC1 orthologous genes in the NCBI database. The amino acid sequence alignment of the CrPEPC1 orthologous genes was created by ClustalW (http:// www.genome.jp/tools/clustalw/). The phylogenetic tree of the CrPEPC1 orthologous genes is presented in Supplementary Fig. 4. As shown in the figure, CrPEPC1 was corresponded with PEPC from algae. All listed CrPEPC1 orthologous genes contain the PEPC function domain. The predicted subcellular location of CrPEPC1 (by Euk-mPLoc 2.0) is the cytoplasm (http://www.csbio.sjtu.edu.cn/bioinf/eukmulti-2/). CrPEPC1 knockdown increases lipid content in C. reinhardtii We examined the effects of CrPEPC1 knockdown on the lipid content in C. reinhardtii to determine the relationship between CrPEPC1 expression and lipid

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Fig. 1 Biomass, lipid content, and TAG level of CrPEPC1 RNAi transgenic Chlamydomonas reinhardtii. Maa7-4(10,19), pMaa7IR/XIR transgenic algal strains; PEPC1-RNAi-6(15,89), pMaa7IR/CrPEPC1IR transgenic algal strains

accumulation. Based on the CrPEPC1 (g16646) sequences retrieved from the Phytozome V9.1 C. reinhardtii database, we designed primers to amplify the fragment of the coding region of CrPEPC1. The DNA fragment was subcloned and then used to generate the CrPEPC1 RNAi construct pMaa7IR/ CrPEPC1IR. More than 120 positive transformants were obtained after transforming the silencing construct into C. reinhardtii CC425. We selected three transgenic algae to measure lipid content by the Nile Red florescence method. One of these algae was used to detect TAG level by GC/MS. The mRNA expression of CrPEPC1 was evaluated by real-time PCR.

Strains transformed with the vector pMaa7IR/XIR were used as controls. Analysis of the transgenic algae revealed that lipid content increased by 20–39 % after 6 days of cultivation. Meanwhile, the TAG level of the transgenic strain PEPC-RNAI-6 increased by 20 % compared with that of the control (Fig. 1; Table 1). We analyzed the abundance of target gene-specific mRNA in the transgenic algae by using real-time PCR to evaluate the effectiveness of our RNAi constructs. The mRNA expression of CrPEPC1 decreased by 74–98 % (Fig. 2a), indicating that the constructs can facilitate high-efficiency silencing. PEPC activity of the transgenic strains was tested. PEPC activity of the transgenic CrPEPC1 RNAi strains decreased by 39–49.5 % compared with those of the pMaa7IR/XIR transgenic strains (Fig. 2b). Subsequently, the mRNA expression levels of DGAT2 (Cre12.g557750) and PAP2 (Cre05.g240000), which are directly related to lipid synthesis, were measured in the transgenic CrPEPC1 RNAi strains. The mRNA expression levels of DGAT2 and PAP2 increased in the transgenic CrPEPC1 RNAi strains (Fig. 3). These results indicates that CrPEPC1 silencing decreases enzyme activities, thereby slowing down cellular TCA and indirectly partitioning photosynthetic carbon into fatty acid and lipid synthesis by increasing DGAT2 and PAP2 gene expression. Similar results were obtained in Nile Red staining; microscopic analysis revealed the presence of a large amount of oil droplets with yellow fluorescence in the CrPEPC1 RNAi transgenic algae than in the pMaa7IR/XIR transgenic algae (Fig. 4). These data indicate that the regulation of CrPEPC1 gene expression can increase cell lipid content despite the different metabolic pathways. CrPEPC1 over-expression reduces lipid content in C. reinhardtii CrPEPC1 knockdown increased lipid content. This result suggests that the expression of this gene affects triglyceride biosynthesis in C. reinhardtii. Thus, we examined whether or not CrPEPC1 over-expression reduces the lipid content in C. reinhardtii. The pCAMPEPC1 vector, which expresses CrPEPC1 from the CAMV 35S promoter, was introduced into C. reinhardtii. The lipid content and growth rate of three randomly selected transgenic algae were detected in each transgenic algae line. CrPEPC1 over-expression

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Biotechnol Lett Table 1 Fatty acids and TAG contents (% cell dry weight) in transgenic strains with silenced or overexpressed CrPEPC1 as analyzed by GC/MS Maa7-4

PEPC1-RNAi-6 Pcambia-2

16:0

0.33 ± 0.06

0.51 ± 0.01

16:147

0.08

16:149 16:2

0.05

pCAMPEPC1-12 0.46 ± 0.02

0.11

0.10 ± 0.01

0.01

0.00

0.05

0.07

0.57 ± 0.08 0.05 ± 0.01

16:4

0.44 ± 0.02

0.43 ± 0.01

0.59 ± 0.02

0.12 ± 0.01

18:0

0.05

0.10

0.05

0.46 ± 0.05

0.49 ± 0.01

0.22 ± 0.01

18:1

0.13 ± 0.01

18:2

0.39 ± 0.02

0.45 ± 0.01

18:345,9,12

0.52 ± 0.02

0.51 ± 0.02

18:349,12,15

0.77 ± 0.10

1.16 ± 0.09

1.11 ± 0.09

0.45 ± 0.07

TAG

2.77 ± 0.23

3.33 ± 0.14

2.87 ± 0.15

1.87 ± 0.23

Maa7-4, pMaa7IR/XIR transgenic algal strain number 4; PEPC1-RNAi-6, pMaa7IR/CrPEPC1IR transgenic algal strain number 6; pCAMBIA-2, pMCAMBIA1302 transgenic algal strain number 2; pCAMPEPC1-12, pCAMPEPC1 transgenic algal strain number 12

A

B

Fig. 2 Comparison of the mRNA abundance and enzyme activity of CrPEPC1 in transgenic Chlamydomonas reinhardtii. a mRNA abundance of CrPEPC1 in transgenic C. reinhardtii, b PEPC activity in CrPEPC1 RNAi transgenic C. reinhardtii. Maa7-4(10,19), pMaa7IR/XIR transgenic algal strains; PEPC1RNAi-6(15,89), pMaa7IR/CrPEPC1IR transgenic algal strains. Statistical analysis was performed using SPSS statistical software. Significance is considered at *p \ 0.05, **p \ 0.01

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Fig. 3 mRNA abundance of acyl-CoA:diacylglycerol acyltransferase (DGAT2) and phosphatidate phosphatase (PAP2) genes in CrPEPC1 RNAi transgenic algal strains. Maa74(10,19), pMaa7IR/XIR transgenic algal strains; PEPC1RNAi-6(15,89), pMaa7IR/CrPEPC1IR transgenic algal strains

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Maa7-4

PEPC1-RNAi-6

A

B

Fig. 4 Microscopic observations of CrPEPC1 RNAi transgenic C. reinhardtii. After 4 days of cultivation in HSM medium, a large amount of oil droplets of CrPEPC1 RNAi transgenic algae was found. Maa7-4, pMaa7IR/XIR transgenic algal strain number 4; PEPC1-RNAi-6, pMaa7IR/CrPEPC1IR transgenic algal strain number 6. Scale bar 10 lm

increased the growth rate of the algae in the early stages from Day 2 to Day 5. CrPEPC1 over-expression in the transgenic strain pCAMPEPC1-12 decreased lipid content and decreased TAG level by 37 % compared with that of the control (Fig. 5; Table 1). Compared with pCAMBIA1302 transgenic stains, the mRNA expression levels of CrPEPC1 increased by 300–325 %, whereas the enzyme activities of PEPC increased by 157–184 % (Fig. 6). In summary, CrPEPC1 over-expression caused photosynthetic carbon flux to the TCA but not to fatty acid synthesis. As a result, lipid synthesis was decreased in the algal cells. Enhancement of PEPC activity accelerated the activity of the TCA and thus increased ATP synthesis, as demonstrated in the increased early growth rates of transgenic algal strains. Nile Red dye staining also showed a decrease in lipid content (Fig. 7). CrPEPC1 expression in Escherichia coli BL21 and enzyme activity detection in vivo The plasmid pGEX-6p-1-PEPC1 was constructed to express CrPEPC1 in E. coli and evaluate enzyme

C

Fig. 5 Biomass, lipid content, and TAG level of CrPEPC1overexpressing transgenic algae in HSM medium. a Growth curve of CrPEPC1 transgenic algae, b lipid content detected by Nile Red florescence method of CrPEPC1 transgenic algae, c TAG level detected by GC/MS after 6 days of cultivation. pCAMBIA-2(8,16), pCAMBIA1302 transgenic algal strains; pCAMPEPC12(35,59), pCAMPEPC1 transgenic algal strains

activities. The recombinant vector was transformed into the E. coli BL21 strain. Transformants were grown in lysogeny broth medium and induced with 1 mM IPTG. The supernatant fraction of the denatured cells was loaded onto a 15 % SDS-PAGE gel. A glutathione S-transferase (GST)-CrPEPC1 protein band of *120 kDa was observed (Fig. 8a). The fusion proteins were purified with columns followed by enzyme activity assay (Fig. 8b, c). The enzyme

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A

pCAMBIA-2

pCAMPEPC1-12

B

Fig. 6 Comparison of the mRNA abundance and enzyme activity of CrPEPC1 in transgenic C. reinhardtii. a mRNA abundance of CrPEPC1 transgenic algae, b PEPC enzyme activity of CrPEPC1 transgenic algae pCAMBIA-2(8,16), pMCAMBIA1302 transgenic algal strains; pCAMPEPC12(35,59), pCAMPEPC1 transgenic algal strains. Statistical analysis was performed using SPSS statistical software. Significance is considered at *p \ 0.05, **p \ 0.01

activity of PEPC increased by 27- to 31-fold compared with that of the control (Fig. 8c). This behavior indicates that the cloned CrPEPC1 gene exhibits biologic activities.

Discussion Metabolic networks of photosynthetic eukaryotic organisms are complicated by the interaction between the synthesis and degradation of carbohydrates, proteins, and lipids. Hence, the regulation of a main carbon metabolic pathway can affect other metabolic pathways. Substrates for fatty acid synthesis can be improved by regulating metabolic pathways other than lipid synthesis to enhance lipid production. Studies on the relationships between carbon flux and lipid accumulation have focused on Arabidopsis thaliana and oil crops. So far, the genes involved include ACCase, sn-glycerol-3-phosphate dehydrogenase, pyruvate dehydrogenase kinase, pyruvate kinase

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Fig. 7 Lipid content in transgenic algal line as detected by Nile Red staining. After 4 days of cultivation in HSM medium, a small amount of oil droplets of CrPEPC1 transgenic algae was found. pCAMBIA-2, pMCAMBIA1302 transgenic algal strain number 2; pCAMPEPC1-12, pCAMPEPC1 transgenic algal strain number 12. Scale bar 10 lm

complex, and D-glucose-6-phosphate dehydrogenase. Ratcliffe and Shachar-Hill (2006) labeled carbon with an isotope in rape seed development and found that the main sources of carbon flux are cane sugar (sucrose) and glucose during rape seed development. Photosynthetic carbon flux can be divided into four main parts: 6.1 % was used to synthesize OAA via PEPC-catalyzed PEP in the glycolytic pathway; 24 % entered the TCA pathway via pyruvate and acetylCoA produced from PEP; 24 % was used to synthesize amino acids via the intermediate products glyceraldehyde-3P, glycerate-3P, and PEP; and the remaining portion was used for lipid production via acetyl-CoA. Therefore, carbon can flow easily into lipid synthesis by directly or indirectly inhibiting PEPC activity, the TCA pathway, and protein synthesis. In the present study, two classes of PEPC complex, PEPC1 and PEPC2, were investigated. PEPC1 is a homotetramer composed of the p109 subunit (4 of 974 amino acids), whereas PEPC2 is a heteropolymer composed of the p109 and p131 subunits (Rivoal et al.

Biotechnol Lett Fig. 8 Expression of CrPEPC1 in E. coli BL21 and in vitro enzyme activity assay. After being induced by IPTG and cultivated in 0, 2, 4, and 6 h, the total protein was harvested and run on SDS–PAGE. a Expression of CrPEPC1 in E. coli BL21, b purified GST-CrPEPC1, c in vitro enzyme activity assay of GST-CrPEPC1

A

B GST-CrPEPC1

GST

M

CK PEPC1

6h 4h 2h 0h 6h 4h 2h 0h M 97.2KDa 66.4KDa

97.4KDa

44.3KDa

42.7KDa

66.2KDa

29.0KDa

31.0KDa

20.1KDa

14.3KDa

14.4KDa

C

1998; Mamedov et al. 2005). The p109 subunit is coded by CrPEPC1 (g16646), whereas the p131 subunit is coded by CrPEPC2 (Cre03.g171950). In this study, we cloned the p109 subunit, the CrPEPC1 gene. Results indicated that CrPEPC1 knockdown or over-expression changed the levels and activities of PEPC1 and PEPC2, and indirectly modulated the lipid accumulation in Chlamydomonas. CrPEPC1 knockdown created the artificial blockage of the TCA pathway and caused the carbon flux to enter the oil accumulation in C. reinhardtii. Further studies are required to elucidate the mechanisms underlying the exact signals triggering such switches. Our findings suggested that oil production can be increased by suppressing CrPEPC1 expression in microalgae. Acknowledgments This work was supported by the National Natural Science Foundation of China (31160050, 31360051, 31000117), the Major Technology Project of Hainan (ZDZX2013023-1), the National Nonprofit Institute Research Grants (CATAS-ITBB110507, 130305), the Funds of Hainan Engineering and Technological Research (GCZX2011006, GCZX2012004, GCZX2013004), and the Natural Science Foundation of Hainan Province (313077).

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Moellering ER, Ouyang YX, Mamedov TG, Chollet R (2007) The two divergent PEP-carboxylase catalytic subunits in the green microalga Chlamydomonas reinhardtii respond reversibly to inorganic-N supply and co-exist in the highmolecular-mass, hetero-oligomeric class-2 PEPC complex. FEBS Lett 581:4871–4876 O’Leary B, Park J, Plaxton WC (2011) The remarkable diversity of plant PEPC (phosphoenolpyruvate carboxylase): recent insights into the physiological functions and post-translational controls of non-photosynthetic PEPCs. Biochem J 436:15–34 Ratcliffe RG, Shachar-Hill Y (2006) Measuring multiple fluxes through plant metabolic networks. Plant J 45:490–511 Rivoal J, Plaxton WC, Turpin DH (1998) Purification and characterization of high- and low-molecular-mass isoforms of phosphoenolpyruvate carboxylase from Chlamydomonas reinhardtii. Kinetic, structural and immunological evidence that the green algal enzyme is distinct from the prokaryotic and higher plant enzyme. Biochem J 331:201–209