Proteomic Analysis of Lipid Accumulation in Chlorella

School of Chemical Engineering, Xiangtan University, Xiangtan 411105, P.R. China. Energy Fuels , 2013, 27 (7), pp 4031–4040. DOI: 10.1021/ef4000177...
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Proteomic Analysis of Lipid Accumulation in Chlorella protothecoides Cells by Heterotrophic N Deprivation Coupling Cultivation Yuqin Li,* Zhengqiu Yuan, Jinxiu Mu, Di Chen, and Bo Feng* School of Chemical Engineering, Xiangtan University, Xiangtan 411105, P.R. China ABSTRACT: The heterotrophic nitrogen (N) deprivation (HND) was the first employed to culture Chlorella protothecoides for microalgal lipids, which are regarded as one of the most promising feedstocks for biodiesel production. First, C. protothecoides was cultivated heterotrophically to achieve high biomass, and the broth was then transferred to N deprivation environment for lipid accumulation. This study aims to investigate proteomic changes in C. protothecoides cells and identify the molecular pathways responsible for lipid storage with HND. Approximately 72% of biomass (13.89 g/L) accumulated as lipids after 240 h, which equates to a lipid productivity of 10.0 g/L. This result represents an increase of 79.5% compared with the lipid yield from the simple heterotrophic mode. Furthermore, 33 altered proteins in HND-cultured algal cells were successfully identified, including 13 down-regulated proteins involved in photosynthesis, protein synthesis and folding, gene regulation and β-oxidation of fatty acids; 15 up-regulated proteins related to carbohydrate metabolism, stress response and defense, amino acid biosynthesis and secondary metabolite biosynthesis; and 5 hypothetical proteins. Analysis using the Kyoto encyclopedia of genes and genomes showed that the carbohydrate metabolism and inhibition of fatty acid catabolism are major routes for lipid accumulation in algal cells. Our results proved that the combination of heterotrophism and N deprivation can increase lipid productivity for algal-based biodiesel. In future studies, further functional analysis of these altered proteins would help elucidate the complicated relationship between cell growth and lipid accumulation in microalgae.

1. INTRODUCTION Currently, new energy sources are being investigated because of the exhausted stocks of fossil fuels and atmospheric pollution. As one of the promising energy sources, biodiesel is a renewable, biodegradable, nontoxic form of bioenergy.1 However, conventional biodiesel feedstocks, such as soybean, palm, jatropha, corn and canola, cause controversial competition with food supply and agricultural land.2 Recently, microalgae have received considerable attention as a promising feedstock for biodiesel production because of their relatively fast growth and high oil content.3 However, microalgae biodiesel production is not economically feasible because rapid-growing microalgal cells contain less oil, whereas those cells with high oil content show low growth ability.4 Efficient biomass and high lipid content can lead to high lipid yield. Numerous reports showed that cell biomass or lipid content could be efficiently increased by growth regimes and conditions, such as heterotrophism, nutritional imbalances of nitrogen (N), phosphate, and silicon, and extreme environmental stress conditions (i.e., high salt, high iron, high light, and low temperature).5−14 Heterotrophism is a cost-effective and relatively simple cultivation regime for numerous microalgal species. This cultivation mode is supported by many previous studies with Chlorella and other microalgae species.4,5,15−18 These studies achieved high lipid content (50.2% to 53.3%) but relatively low biomass in C. protothecoides cells, which resulted in low lipid yield. Wei et al. obtained high biomass (15.8 g/L) in C. protothecoides with cassava starch hydrolysate as carbon source; however, the final lipid yield was not satisfactory because the corresponding lipid content was low. These investigations do not have a positive balance between biomass and lipid content. Thus, the final lipid yields do not meet the requirements of sustainable biodiesel. © XXXX American Chemical Society

Recently, a two-step culture regime has been developed to produce high cell biomass with high intracellular lipid content. For example, a photosynthesis-fermentation model was adopted by merging the positive aspects of autotrophs and heterotrophs, where C. protothecoides was first grown autotrophically for CO2 fixation and then metabolized heterotrophically for oil accumulation. 19 Chlorella sp. BUM11008 was cultivated under a 12/12 h light/dark photoperiod to achieve high cellular biomass and then transferred to nutrient-deprived medium to promote intracellular lipid accumulation.20 Mujtaba et al. also developed a two-stage process for lipid accumulation by C. vulgaris: growth under nutrient-rich conditions and cultivation under N starvation.21 Approximately 53% of dry cell weight consisted of lipids after 24 h, representing a lipid productivity of 77.1 mg/ L/d. Recently, Fan et al. have investigated a novel two-step culture model comprising sequential heterotrophy-dilutionphotoinduction to increase algal biomass and lipid production.22 Using this model, the lipid content was increased by 84.57%, 70.65%, and 121.59% within 24 h photoinduction for C. vulgaris, C. pyrenoidosa, and C. ellipsoidea, respectively. These findings demonstrate that two-step culture systems can obtain high biomass with high lipid content. However, the environmental adaptability of microalgae from dark to light and electric energy consumption cost should be considered. Therefore, the exploitation of a cost-effective and feasible culture regime for high biomass and mass algal lipid production is particularly significant. Received: January 5, 2013 Revised: May 15, 2013

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Cell Observations via Transmission Electron Microscopy. After three to four washes with distilled water followed by 2 min of centrifugation at 15 rpm in ambient temperature, a mixed culture of Chlorella was suspended in a solution of 3.0% glutaraldehyde in distilled water and then incubated at 4 °C for 2 h with gentle mixing every 10 min. The glutaraldehyde-treated cells were centrifuged and then suspended in 0.1 M phosphate buffer. The cells were subsequently postfixed in 1% osmium tetraoxide for 90 min at 4 °C to a final concentration of 2% (v/v) and then buffered with 0.1 M phosphate. The samples were then dehydrated by incubation in a graded series of 30%, 50%, 70%, 80%, 90%, and 100% ethanol for 10 min each. The ethanol was then replaced by an embedding solution consisting of propylene oxide and epoxy resin (2:1, v/v). After 20 min of incubation, the dehydrated sample was replaced with a fresh embedding solution and incubated for an additional 20 min. Propylene oxide was removed via 4 h of vacuum pumping. The final samples in the embedding molds were placed in a drying oven at 70 °C and then heated gradually to polymerize the epoxy thoroughly. After ultrathin sectioning, the samples were incubated for three successive 20 min time periods in uranyl acetate for staining, incubated for 10 min in lead citrate for poststaining, and finally heated to 60 °C for drying. The stained and dried samples were positioned on grids and visualized via transmission electron microscopy (TEM; JEOL JEM-1230, Japan). Proteomics Analysis. Protein Extraction and Purification of Algal Cells. After medium removal, the cultured cells were collected and washed thrice in ice-cold phosphate-buffered saline buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4; pH 7.4). Then, algal cells were directly disrupted in 1 mL of lysis buffer (7 M urea, 2% CHAPS, 2 M thiourea, and 20 mM Tris; pH 8.8) containing 1 mM phenylmethylsulfonyl fluoride protease inhibitors. The mixture was subjected to ultrasonic treatment using a sonicator (Ningbo Scientz Biotechnology Co., Ltd., JY96-IIN, China) to break DNA and RNA. After pelleting the insoluble material by centrifugation at 18,000 × g for 20 min at 4 °C, the supernatant was collected. The protein samples were cleaned with Clean-up kit according to the manufacturer’s instructions. The purified supernatant was added into 1 mL of ice-cold acetone to precipitate protein at 4 °C overnight. The precipitated protein clumps were separated by centrifugation at 4 °C for 20 min and then mixed with rehydration buffer stock (7 M urea, 2 M thiourea, 4% CHAPS). Finally, the protein samples were quantified with 2D Quant Kit according to the manufacturer’s instructions. Two-Dimensional Gel Electrophoresis. Onto each gel, 250 μg of protein was loaded and separated using two-dimensional (2-D) gel electrophoresis (2-DE). First-dimensional gel electrophoresis was carried out using 24 cm immobilized pH gradient (IPG) strips with a linear pH 4 to 7 gradient (BioRad) and a GE ETTAN IPGPHOR 3 (GE Healthcare) set at 20 °C. The strips were rehydrated in a solution (2 M thiourea, 40% w/v CHAPS, 20 mM w/v DTT, 0.5% v/v IPG buffer pH 4 to 7, 0.002% w/v bromophenol blue, and 8 M urea). Isoelectric focusing was performed by stepwise increase of the voltage as follows: in the first step, voltage was held at 300 V for 30 min; in the second step, voltage was quickly increased from 300 to 700 V within 60 min; in the third step, voltage was linearly increased from 700 V to 1,500 V within 5 h; in the last step, voltage was maintained at 9,000 V for 10 h. After equilibrating and incubating in buffer I [50 mM TrisHCl, pH 8.8, 6 M urea, 2% sodium dodecyl sulfate (SDS), 30% glycerol, and 1% dithiothreitol (DTT)] for 20 min and buffer II (50 mM Tris-HCl, pH 8.8, 6 M urea, 2% SDS, 30% glycerol, and 2.5% indole-3-acetic acid) for 15 min, the strips were ready to be applied to the 2-D gels for 12.5% SDS-polyacrylamide gel electrophoresis (SDSPAGE). Separation was performed in an Ettan Dalt system (GE Healthcare) at 15 mA/gel for 45 min and at 30 mA/gel for 4.5 h until the bromophenol blue dye front reached the bottom of the gel. Each sample was run in duplicate. 2-DE Image Capture and Analysis. The 2-D gel images were scanned using a Typhoon laser scanner (GE Healthcare, Uppsala, Sweden) at a resolution of 150 dpi and analyzed using ImageMaster 2D platinum 5.0 (GE Healthcare, Milwaukee, WI, USA). Spot detection, background subtraction, and spot quantitation were performed on 16-bit TIEF images acquired with a scanning

In this study, C. protothecoides was adopted to verify the feasibility of a novel heterotrophic N deprivation (HND) coupling cultivation strategy for efficient biomass and lipid production. Chlorella was first cultivated heterotrophically to achieve high cell biomass, and then algal cells in the late exponential phase were collected and reinoculated in Ndeprived medium for lipid accumulation. Using this strategy, we found that 72% of biomass (13.89 g/L) accumulated as lipids after 240 h, corresponding to a lipid productivity of 10.0 g/L. This result shows an increase of 79.5%, compared with the lipid yield from simple heterotrophic mode. Analysis using the Kyoto encyclopedia of genes and genomes (KEGG) showed that the carbohydrate metabolism and inhibition of fatty acid catabolism are major routes for lipid accumulation in algal cells by HND. This study is the first to report on lipid accumulation ability and mechanism in algal cells with HND via an integrated approach composed of biochemical, ultrastructural, and proteomic analyses.

2. MATERIALS AND METHODS 2.1. Algal Strain, Medium, and Culture Conditions. The green microalga C. protothecoides CS-41 was purchased from the CSIRO Marine Laboratory, Hobart, Australia. For routine operations, the strain was activated and inoculated into 250 mL Erlenmeyer flasks with 100 mL of basal culture medium (BCM) composed of the following (per L): 1.25 g of KH2PO4, 1 g of MgSO4•7H2O, 83.75 mg of CaCl2, 49.8 mg of FeSO4•7H2O, 15.7 mg of CuSO4•5H2O, 38.2 mg of ZnSO4•7H2O, 0.5 g of EDTA, 114.2 mg of boron, 14.4 mg of MnCl2•4H2O, 7.1 mg of MoO3, 4.9 mg of CoNO3•6H2O, 3 of g urea, and 30 g of glucose. The HND coupling cultivation process was adopted to culture the microalga C. protothecoides, and the cultivation process was carried out by two-stages: (1) Heterotrophic phase: 1 mL of the activated seed liquid was inoculated into a 250 mL Erlenmeyer flask with 100 mL of BCM medium, and the culture was then incubated in an incubator shaker with 160 rpm/min at 28 °C without illumination until the cells reached the late logarithmic phase. (2) N-Deprivation phase: The cultured cells in late logarithmic growth phase (168 h) were collected and centrifuged at 8000 g for 10 min to remove the supernatant. The algal pellets were then washed twice with their corresponding Ndeprivation medium and reinoculated into fresh N-deprived BCM-N medium containing the same components as BCM, except for 0, 0.15, 0.30, 0.45, and 0.60 g/L urea instead of 3 g/L urea. Subsequently, the cultures were incubated for another 72 h in an orbital shaker with 160 rpm/min at 28 °C without light exposure. Then, the algal cells were collected and treated with biological indexes analysis. 2.2. Methods. Biochemical Analysis. Biomass. For biomass determination, 10 mL of cultured fluid was transferred to a preweighed centrifuge tube and centrifuged at 8,000 g for 10 min. After rinsing several times with distilled water, the algal slurry was dried at 50 °C to constant weight in a sirocco-blasting drying trunk and then cooled to room temperature. The biomass was determined using an electronic balance. Total Lipid. The dried cell biomass was blended with 0.5 mL of distilled water and then disrupted using a microwave oven at high temperature (approximately 100 °C and 2450 MHz) for 5 min. Then, total lipid extraction was performed by mixing 3 mL of chloroform/ methanol (2:1, v/v). The mixtures were shaken for 20 min using a mixer and then centrifuged at 10,000 rpm for 10 min. All the chloroform phases were collected together, evaporated, and dried to constant weight under vacuum conditions. The extracted total lipid weight was determined by the weighing method with an electronic scale. Finally, the total lipid yield (g/L) was calculated by the total lipid weight/the volume of culture liquid. All biological assays were carried out in triplicate, and mean values and standard deviations were calculated. B

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Figure 1. Effects of HND regime on the biomass and lipid content of C. protothecoides. The HND was carried out by two-step cultivation: First, the algal cells were heterotrophically cultured for 7 days (the late of logarithmic phase), shown in heterotrophic phase; second, the algal cells in the late of logarithmic phase were reinduced into N deficiency medium (0, 0.15, 0.30, 0.45, 0.60 g/L of urea) for 3 days, shown in N deprivation phase. Error bars represent the mean ± standard deviation (SD) of three independent biological replicates. densitometer. The computer analysis allowed automatic detection and quantification of protein spots as well as matching between the control (heterotrophic cultivation) and experimental (coupling cultivation) gels. Only proteins with significant and reproducible changes were considered to be differentially accumulated proteins. Significant differences of the protein spots were evaluated by Student’s t test; p < 0.05 was considered to indicate statistical significance. In-Gel Digestion of Proteins. The changed protein spots were manually excised from the gel and subjected to in-gel digestion. Matrix-assisted laser desorption/ionization (MALDI) samples of the extracted peptides were prepared following the method described by Kurian et al.23 Protein Identification. Protein identification was carried out on MALDI-TOF-TOF-MS (Autoflex speedTM, Bruker Dalton, Bremen, Germany) in the positive ion reflectron mode. Equal volumes of trypsinised samples (0.5 μL) and matrix solution (0.5 μL) containing 5 mg/mL α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich Fluka, St. Louis, MO) were prepared in 50% acetonitrile, 0.1% v/v trifluoroacetic acid, and 2% w/v ammonium citrate. The sample/matrix mixture was spotted onto a 96-well MALDI-TOF MS target plate. We used a protein molecular mass range of 6 kDa to 200 kDa and a mass tolerance of 100 ppm for internal calibration. For protein identification, we performed database searching against the NCBInr database using the MASCOT program and the MASCOT search engine with the following parameters: (1) molecular weight (MW) of protein; (2) isoelectric point (pI); (3) one missed cleavage; (4) global modification, carbamidomethyl; and (5) variable modifications, oxidation (M). The expectation value (chance of misidentification) is less than 0.05.

15.08 g/L, which represents 2.9- to 4.1-fold higher than those reported in the literature.4,5,15,16 However, the lipid content was only 37% of dry cell biomass. After the heterotrophic phase, the algal biomass in the late logarithmic stage was collected and transferred into N-deficient medium (0, 0.15, 0.30, 0.45, and 0.60 g/L urea) for continual cultivation. After 72 h (3 d), the biomass yields varied with different levels of urea concentration. In general, with the concentration ranging from 0 to 0.60 g/L, the biomass of C. protothecoides increased with the increases of urea concentration. As shown in Figure 1A, the maximum biomass was 17.681 g/L with 0.60 g/L urea feeding. However, when N was absolutely absent (0 g/L), the biomass decreased to 13.89 g/L. The results imply that N-sufficient and Ndeficient conditions promote different cell behavior, and this phenomenon is also consistent the reports of refs 24 and25 who cultivated marine microalga Ellipsoid ion sp. using urea as the nitrogen source. Lipid content was also significantly different with the level of urea concentration. As shown in Figure 1B, the intracellular lipid content increased with decreasing urea concentrations. When algal cells were fed with 0.15 g/L to 0.60 g/L urea, the lipid content occupied 41% to 58% of dry biomass at the end of the N deprivation phase. These values were higher than the 37% from the heterotrophic phase with 3 g/L urea. The absence of urea in the medium resulted in a maximum lipid content of 72% of dry cell biomass. These observations suggest that the metabolism of lipids can be controlled by regulating N concentration. The absence of urea is considered an appropriate strategy to achieve high lipid yield. 3.2. Morphology of C. protothecoides Cells with HND. To investigate the morphological and cytological changes by HND regime, C. protothecoides cells were visualized via TEM. The algal cells were spherical, ellipsoidal, or ovoid with a diameter range of 2.0 to 12.0 μm (Figure 2A). Cellular cleavage and proliferation significantly increased during the heterotrophic phase. First, algal cell proliferation formed several autospores by mitosis. The autospores grew and expanded, resulting in cell wall breakage of the brood cell. Then, several autospores broke away from the brood cell and became vegetative cells (Figure 2B). This process is constantly repeated until the late logarithmic stage. As shown in Figure 2C, lipid and starch droplets began to form inside algal cells (late log phase). However, the number of starch droplets was slightly

3. RESULTS 3.1. Effects of HND Regime on the Biomass and Total Lipid Production of C. protothecoides. High cell biomass was easily obtained by adding sufficient nutrition ingredients (e.g., carbon and N) into the medium, but it was useless for lipid accumulation. Many extreme environmental stress conditions, including nutrition limitation, were tested to promote lipid accumulation, such as N and phosphate deprivation. Nitrogen is relatively inexpensive and can be favorably manipulated compared with other nutrient components. Thus, accumulating intracellular lipid for biodiesel production is critical.8 Based on the growth and lipid accumulation characteristics of C. protothecoides, a coupling mode (HND) was developed. As shown in Figure 1A, after 168 h (7 d) of heterotrophic phase, high biomass was obtained by C

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component), H26 and H30 (sugar nucleotide epimerase), and H31 and H40 [fructose-1,6-bisphosphate (FB) aldolase]. These proteins represent different modifications of the same gene product or isoforms of the same protein family. Compared with the control group, three protein spots (H25, H30, and H34) were newly induced proteins, and one protein spot (L48) disappeared from the algal cells after N deficiency cultivation (Figure 3E and Table 1). The identified proteins participate in nine metabolic activities: photosynthesis, protein biosynthesis and folding, defense and stress responses, gene regulation, amino acid metabolism, carbohydrate metabolism, fatty acid catabolism, secondary metabolite biosynthesis, and hypothetical proteins. The down-regulated proteins were mainly involved in photosynthesis, protein biosynthesis and folding, gene regulation, and lipid metabolism (β-oxidation of fatty acid). Among the down-regulated proteins, seven proteins were involved in photosynthesis (L29, L31, L32, L34, L39, L46, and L67), implying that photosynthesis and electron transport were largely inhibited in Chlorella cells after N deficiency. Four proteins (L41, L76, L78, and L79) participated in protein synthesis and folding, comprising PPIase, ribosomal protein L15, and two ribosomal proteins S12, which were also observed to be significantly down-regulated. The results suggest a typical response of cell growth inhibition by N deficiency. One protein (L48) on gene regulation disappeared in BCM-N Chlorella cells. Acyl-CoA dehydrogenase (L70) involved in lipid metabolism-β-oxidation was down-regulated, indicating a promising strategy for intracellular lipid accumulation. Two hypothetical protein proteins (L1 and L11) were also downregulated in N-deficient cells. The up-regulated proteins fell into five major biological process groups (Table 1). Seven proteins (H7, H12, H26, H30, H31, H40, and H47) were implicated in carbohydrate metabolism. Among these proteins, H31, H40, and H47 were involved in pyruvate synthesis, which is the final output of glycolysis. This result suggests that N deficiency enhances carbohydrate metabolism activity, resulting in more pyruvate. The up-regulated transketolase (H2) participates in the pentose phosphate pathway (PPP). Two up-regulated proteins (H33 and H48) were involved in amino acid metabolism. The results suggest that cell growth was decreased by N deficiency. However, amino acids and proteins are still needed to form valuable enzymes in fatty acid de novo biosynthesis and to resist the adverse situation, respectively. H10, H11, H19, and H38 are stress response and defense proteins. Their enhanced expression accelerates cell recovery from nutrition limitation conditions. Another significantly up-regulated protein (H44) is CPX1, which belongs to secondary metabolite biosynthesis. These altered proteins are likely to provide new insights about lipid accumulation in C. protothecoides cells after N deficiency. The NCBI accession number, protein name and species, mascot score, numbers of peptides matched, and coverage (percentage of predicted protein sequence covered by matched peptides) are listed in Table 1. A representative MALDI-TOF/TOF MS peptide mass fingerprint spectrum for spot H7 (NAD-dependent epimerase/dehydratase) is shown in Figure 4.

Figure 2. Transmission electron microscopy images of C. protothecoides cells. (A) Microalgae cellular morphology; (B) Microalgal cell proliferation mode during the heterotrophic phase; (C) C. protothecoides cells grown in BCM for 7 days; (D) C. protothecoides cells grown in BCM-N for 3 days. CW: Cell wall; L: lipid droplet; St: starch droplet.

greater than that of lipid drops. This result indicates that starch synthesis mainly occurred through glucose metabolism in the heterotrophic phase, with a small amount of oil droplet formation. When the algal cells were subjected to N deficiency induction, an obvious increase in the amount of oil bodies was observed, and this trend continued until oil bodies occupied most of the cell area (Figure 2D). By contrast, starch granules decreased rapidly within 3 d after N starvation. Distinguishing the nucleus was also difficult because of its dramatic disappearance in the N starvation induction process. This result confirms that lipids accumulated in C. protothecoides with HND. 3.3. Identification of Differentially Expressed Proteins in Response to HND in C. protothecoides. To investigate the protein profile changes in algal cells with HND regime, samples of 250 μg protein were used and separated by 2DPAGE. The representative 2-D images are shown in Figures 3A and 3B. The 2-DE images were compared between BCM-N (N-deficient) and BCM (as the reference, N-sufficient) using the PDQuest program to identify variations in protein spots. Thus, the overlap between BCM-N and BCM contains a set of 50 protein spots, presenting more than 3.0-fold changes in abundance (p < 0.05; Figure 3C). A total of 50 differentially expressed protein spots were manually excised from gels and identified using MALDI-TOF/ TOF MS followed by NCBInr database search. Ultimately, 33 differentially expressed protein spots were successfully identified, which included 18 up-regulated proteins and 15 downregulated proteins. In addition, 17 differentially expressed protein spots have not received positive identification. The identified proteins are shown in Figure 3D. A few protein spots were identified as the same protein with different MW and pI values, such as L29, L31, L32, L34, L39, L46, and L67 (lightharvesting chlorophyll-a/b binding protein), L78 and L79 (ribosomal protein), H10 and H19 (chaperonin complex

4. DISCUSSION Microalgae are considered as a promising biological material for biodiesel production because of their relatively fast growth and high lipid content.26,27 However, microalgae-derived biodiesel D

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Figure 3. The 2D gel images from algal cells with molecular weight and pH indicated on the left and head of the 2-DE gels, respectively. All gels were run in triplicate. The protein (250 μg) was applied to 24 cm pH 4−7 IPG dry strips with 12.5% linear vertical SDS-PAGE as the second dimension. The gels were stained with Coomassie blue G-250, and the synthetic gel images were generated using the PDQUEST program. The arrows with numbers on 2D gel indicate the changed proteins, which are further identified by MALDI-TOF/TOF MS. (A) Image of proteins from algal cells grown in BCM; (B) Image of proteins from algal cells grown in BCM-N; (C) The changed 50 protein spots indicated by numbers and arrows; (D) Thirty-three of the proteins are numbered and identified by MALDI-TOF/TOF-MS; (E) Thirty-three of the identified protein spots are enlarged with curve lines. Numbers in the image correspond to the identified proteins described in Table 1. E

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Table 1. Identified Differentially Expressed Proteins after 2D-DIGE Coupled with MALDI-TOF-TOF Mass Spectrometry Analysis in C. protothecoides Cells in Response to HND spot IDa

accession no.b

protein name [species]

L29

gi307106802

L31

gi307106802

L32

gi307106802

L34

gi307106802

L39

gi307103502

L46

gi307103588

L67

gi307103502

L76

gi307108729

L41 L78 L79 L48 L70

gi55976534 gi307106467 gi307106467 gi255629756 gi307108887

H2

gi307106735

light-harvesting chlorophyll-a/b binding protein Lhcb5 [Chlamydomonas incerta] Light-harvesting chlorophyll-a/b binding protein Lhcb5 [Chlamydomonas incerta] light-harvesting chlorophyll-a/b binding protein Lhcb5 [Chlamydomonas incerta] light-harvesting chlorophyll-a/b binding protein Lhcb5 [Chlamydomonas incerta] photosystem II manganese-stabilizing polypeptide (MSP) [Micromonas pusilla_CCMP1545] chlorophyll a/b-binding protein [Coccomyxa subellipsoidea C-169] oxygen-evolving enhancer protein 1 of photosystem II (PSBO) [Chlorella variabilis] peptidyl-prolyl cis−trans isomerase, FKBP-type (PPIase) [Chlamydomonas reinhardtii] 60S ribosomal protein L5 [Triticum aestivum] 40S ribosomal protein S12 [Zea mays] 40S ribosomal protein S12 [Branchiostoma belcheri] zinc finger protein [Medicago truncatula] Acyl-CoA dehydrogenase [CHLREDRAFT_ 118751] transketolase (TRK1) [CHLREDRAFT_141319]

H7

gi307104044

H12

gi307102543

H26

gi384253263

H30

gi384253263

H31

gi255083400

H40

gi307103579

H47

gi145343997

H10

gi307108373

H19

gi307107274

H11

gi6729524

H33

gi307108678

H48

gi307110366

H38 H44

gi307103384 gi75232919

L1

gi307107596

L11

gi303285514

H25

gi307111344

H34

gi307106802

H51

gi307111628

NAD-dependent epimerase/dehydratase (SNE4) [CHLREDRAFT_151368] glucose-1-phosphate adenylyltransferase (STA6) [CHLREDRAFT_136037] sugar nucleotide epimerase (SNE1) [CHLREDRAFT_196952] sugar nucleotide epimerase (SNE4) [CHLREDRAFT_196952] fructose-1,6-bisphosphate aldolase (FBA1) [CHLREDRAFT_152892] fructose-1,6-bisphosphate aldolase (FBA4) [CHLREDRAFT_196304] malate dehydrogenase (MDH1) [CHLREDRAFT_ 137163] chaperonin complex component [Volvox carteri f. nagariensis] chaperonin complex component [Volvox carteri f. nagariensis] cysteine proteinases-like protein [Arabidopsis thaliana] glutamine synthetase (GS) [CHLREDRAFT_ 129468] pyrroline-5-carboxylate reductase (PCR1) [CHLREDRAFT_192364] 2-cys peroxiredoxin (Prx) [Ectocarpus siliculosus] coproporphyrinogen III oxidase (CPX1) [CHLREDRAFT_53583] hypothetical protein CHLNCDRAFT_52074 [Chlorella variabilis] hypothetical protein [Micromonas pusilla CCMP1545] hypothetical protein CHLNCDRAFT_29144 [Chlorella variabilis] hypothetical protein CHLNCDRAFT_35818 [Chlorella variabilis] hypothetical protein CHLNCDRAFT_133673 [Chlorella variabilis]

MW/pIc

mascot scored

matched peptidese

coveragef

ratiog

photosynthesis

29643/5.31

90

2

5

−5.43

photosynthesis

29611/5.28

95

2

5

−6.55

photosynthesis

29623/5.25

90

2

5

−4.94

photosynthesis

29600/5.20

89

1

5

−3.81

photosynthesis

31097/5.28

130

1

5

−3.82

photosynthesis

26685/4.81

123

2

13

−12.43

photosynthesis

31097/5.28

77

1

5

−3.27

protein folding

34699/9.02

77

6

19

−3.97

protein biosynthesis protein biosynthesis protein biosynthesis gene regulation lipid metabolism

22910/5.03 15560/4.80 15558/4.74 7656/9.95 15380/4.62

102 182 286 76 157

2 5 5 5 2

8 30 30 54 15

−4.17 −3.36 −4.91 10000 −25.07

carbohydrate metabolism carbohydrate metabolism carbohydrate metabolism carbohydrate metabolism carbohydrate metabolism carbohydrate metabolism carbohydrate metabolism carbohydrate metabolism stress response

73080/5.59

96

4

7

8.93

93462/5.58

272

23

26

5.12

55647/8.89

96

8

17

15.22

45485/5.55

112

4

7

3.20

45470/5.50

105

4

7

10000

36840/5.91

43

1

2

11.80

41752/6.49

66

2

9

25.37

33997/4.85

90

1

4

4.27

58567/5.97

76

2

4

4.89

stress response

111087/7.8

101

7

8

3.49

stress response

34792/8.93

48

1

3

10.28

amino acid metabolism amino acid metabolism defense metabolism of cofactors and vitamins unclassified

41616/6.17

90

2

5

3.32

28912/7.64

56

1

5

18.34

36491/5.00 44129/6.23

100 86

2 3

6 7

13.57 5.43

37255/5.62

66

1

4

−7.20

unclassified

49119/5.46

48

1

2

−22.60

unclassified

45789/6.04

166

6

13

10000

unclassified

29643/5.31

83

2

5

10000

unclassified

83598/9.31

53

3

4

30.09

function categories

a

Spot ID represents the protein spot number on the 2-D gels (shown in Figure 3D). bAccession numbers according to the NCBI database. Observed MW and pI of protein spot in the 2-D gel. dProtein score greater than 42 is significant (p < 0.05). eNumber of peptides that match the predicted protein sequence. fThe ratio of the protein sequence covered by the matched peptides. gSpot abundance is expressed as the average ratio of intensities of up-regulated (positive values) or down-regulated (negative values) proteins. It is noted that “10000” represented these proteins which disappeared or were newly induced in alga cells after N deprivation. c

F

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Figure 4. A representative MALDI-TOF-MS peptide mass fingerprint obtained for tryptic peptides eluted from 2-D gel spot H7. The x-axis represents mass-to-charge ratio (m/z), whereas the y-axis represents relative abundance. (A) The MALDI-TOF mass spectrum of NAD-dependent epimerase/dehydratase (SNE4) (spot H7); (B) The matched peptide sequences are marked with bold type.

low lipid yield.4 Cell proliferation behavior and lipid accumulation greatly depend on diverse factors, including growth temperature and pH; nutrient deprivation of carbon, N, phosphorus and silicate; growth regime (autotrophic, mixo-

has minimal economic competitive advantage over fossil fuels considering the current energy prices. A major obstacle is that rapidly growing algal cells contain less oil, whereas those algal cells with high oil content show slow growth rate, leading to G

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trophic, or heterotrophic); age of the culture; and microalgae strain.28,29 In this study, a two-step cultivation strategy was developed to achieve maximum amounts of algal biomass and high lipid content. This mode used HND by merging the positive aspects of heterotrophism and nutrient limitation. C. protothecoides was cultured heterotrophically to promote cell biomass and then triggered by nutrient deprivation for lipid accumulation. We obtained favorable results in terms of productivities of the resulting biomass (13.89 g/L) and lipid content (72%) in flask experiments. The final lipid yield was achieved by 10.0 g/L, which was 1.8-fold more than the control group (N-sufficient feeding cells). The biomass and lipid yields were 2- to 2.6-fold and 3.7- to 5.0-fold higher, respectively, than those reported in the literature, as indicated in the text with the same C. protothecoides strain. Moreover, an obvious increase in the amount of oil bodies was observed in algal cells after 3 d of N deprivation, and this trend continued until oil bodies occupied most of the cell area (Figure 2). From the perspective of economics, HND can be considered as a suitable two-step cultivation regime. HND balances biomass and lipid yield and involves only minimal and cost-effective nutrients. Thus, HND has a great significance on the overall economics of biodiesel. However, little is known about the underlying molecular basis of lipid accumulation in response to HND. Proteomics was applied to identify the underlying metabolic pathways in C. protothecoides cells and learn more about the molecular route of lipid accumulation. Compared with the control group, seven proteins (spot L29, L31, L32, L34, L39, L46, and L67) related to photosynthesis were down-regulated by 5.43-, 6.55-, 4.94-, 3.81-, 3.82-, 12.43-, and 3.27-fold, respectively, in the algal cells (Table 1). For example, the light-harvesting chlorophyll a/b-binding protein (LHCB) (spot L46) was barely detectable by 2-D image (Figure 3E). LHCBs (spots L29, L31, L32, L34, L46, and L67) are apoproteins of the light-harvesting complex of photosystem II (PSII), which is normally complexed with chlorophyll and xanthophylls, serving as the antenna complex.30 The downregulation of LHCB is consistent with the report that cells have reduced amounts of light-harvesting complexes in Micractinium pusillum after N deprivation.31 MSP (spot L39) and PSBO (spot L67) have important functions in photosynthesis by controlling O2 evolution and maintaining the stability of PS II.32 The entire cultivation process (BCM or BCM-N) was performed in the dark (no illumination). Thus, the downregulation of proteins associated with photosynthesis may be attributed to its degenerated or damaged function. Given that photosynthesis supplies the energy needs of cell growth and proliferation, its damage undoubtedly leads to a decline in the total biomass yield. In our results, the biomass decreased from 15.08 g/L to 13.89 g/L, complying with the aforementioned assumption. A promising strategy for high lipid content is to inhibit lipid catabolism (e.g., β-oxidation of fatty acid). Acyl CoA dehydrogenase is one of the key enzymes in the β-oxidation of fatty acids. In this study, the expression of acyl CoA dehydrogenase (spot L70) dramatically decreased by 25.07-fold after N deprivation, which significantly enhanced lipid accumulation in algal cells (Figure 5). Aside from reducing the expression of key β-oxidation enzymes, lipid catabolism is also inhibited by knocking some genes of these enzymes.33,34 However, cells rely on lipid catabolism for cellular energy under certain physiological conditions. Thus, cellular growth and proliferation were significantly decreased, indicating that the

Figure 5. Schematic diagram of metabolic pathway of lipid accumulation in C. protothecoides cells with HND regime. Four upregulated proteins, fructose-1,6-bisphosphate aldolase (FBA1, H31), fructose-1,6-bisphosphate aldolase (FBA4, H40), sugar nucleotide epimerase (SNE1, H26), and sugar nucleotide epimerase (SNE4, H30) are involved in glycolysis. Glucose-1-phosphate adenylyltransferase (STA6, H12) is involved in starch synthesis. Transketolase (TRK1, H2) is related to pentose phosphate pathway (PPP). Glutamine synthetase (GS, H33) and pyrroline-5-carboxylate reductase (PCR1, H48) participate in amino acid synthesis. Malate dehydrogenase (MDH1, H47) is indirectly involved in pyruvate accumulation. An upregulated hypothetical protein (H25) participates in glyoxylate cycle. L29, L31, L32, L34, L39, L46, and L67 (light-harvesting complex I protein) are related to photosynthesis and were down-regulated in C. protothecoides cells after N deprivation. Acyl-CoA dehydrogenase (L70) is also down-regulated and involved in lipid catabolism (βoxidation).

biomass decreased slightly (Figure 1A). By contrast, zinc finger protein (spot L48) associated with gene regulation disappeared in BCM-N cells, which may be the key to the regulation of lipid accumulation. Protein synthesis is the most important and complicated process in cells. In the present study, four down-regulated proteins (spots L76, L41, L78, and L79) were categorized into protein folding and synthesis. Among them, three proteins were various individual ribosomal proteins, e.g., ribosomal protein L5 and S12 (Table 1). The down-regulation of ribosomal protein supports the work of Li et al., where the expression abundance of several ribosomal proteins related with protein synthesis decreased in M. pusillum after N deprivation.31 This result proves that the protein energy of cell growth and division was reduced after N deprivation so that the biomass was not significantly increased (Figure 1A). Although the cell growth rate decreased, several enzymes in fatty acid de novo biosynthesis and proteins were still needed to resist the adverse situation. Therefore, some molecular chaperones similar to heat-shock proteins exposed to stress by nutrient limitation were highly expressed in BCM-N cells. Their function is to protect proteins from excessive degradation. For instance, protein spots H10, H11, H19, and H38 acting as molecular H

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dehydrogenase involved in gluconeogenesis. Malate is transported from the mitochondria into the cytosol and subsequently oxidized back to oxaloacetate by this enzyme. Finally, oxaloacetate is converted into phosphoenolpyruvate (PEP).38 The production of PEP is integrated into the glycolysis route and ultimately leads to pyruvate accumulation (Figure 5). As the last energy-generating step in glycolysis, pyruvate is the key intersection in the network of metabolic pathways, such as fatty acid synthesis, TCA cycle, and amino acid metabolism. In the present study, the lipid content dramatically increased, suggesting that amounts of acetyl-coA were generated from accumulated pyruvate. Acetyl-CoA is an important precursor for lipid synthesis. Thus, N deprivation induces carbon flow from carbohydrates into lipid synthesis. Five novel (hypothetical) proteins (spots L1, L11, H25, H34, and H51) have never been reported in proteomic data sets in this paper (Table 1). Despite the similarities to proteins from other organisms, the function of hypothetical proteins is mostly unknown. BLASTP (http://www.ncbi.nlm.nih.gov/BLAST/) was used to score the homology of the hypothetical proteins in the database. The hypothetical protein L11 decreased 1.5-fold after N deprivation and showed homology to transcription antitermination protein (NusG). Aliaga Goltsman et al. reported the involvement of L11 in the chemoautotrophic iron-oxidizing Leptospirillum ferrodiazotrophum.39 Bacterial NusG is a component of the transcription complex and interacts with the termination factor Rho and RNA polymerase. Thus, L11 can function as a transcriptional elongation factor involved in transcription antitermination. In N starvation cells, the hypothetical protein H25 is a newly induced protein that shares 97% homology with isocitrate lyase (ICL). ICL participates in the glyoxylate cycle, which catalyzes the cleavage of isocitrate to succinate and glyoxylate.40 The end products of the bypass can be used for gluconeogenesis and other biosynthetic processes. The reports show that the glyoxylate cycle occurs in bacteria, fungi, plants, and microalgae. The other hypothetical proteins showed lower homology with proteins from proteomic data sets without further discussion. In the future, genetic information and protein database will help reveal the functions of these hypothetical proteins in organisms.

chaperones were up-regulated by 3.49-, 10.28-, 4.89-, and 13.57-fold, respectively. The up-regulation of 2-cys peroxiredoxin (Prx; spot H38) was also reported in Scystosiphon gracilis in response to stress.35 Thus, Prx has the potential to protect Chlorella cells from poor nutrition. Many studies verified that low temperature, drought, and high salinity can lead to the accumulation of cysteine proteinase-like proteins in algal cells.36 In this study, the up-regulation of cysteine proteinase-like protein (spot H11) decreased the damage and denaturalization of proteins caused by N deprivation (Table 1). Nutrient limitation stress was found to affect the abundance of two amino acid biosynthesis enzymes (spots H33 and H48) and one cofactor biosynthesis enzyme (spot H44). Glutamine synthetase (spot H33) has an essential function in N metabolism by catalyzing the condensation of glutamate and ammonia to form glutamine, which can be used to synthesis protein (Figure 5)37 or provide antioxidation protection and promotes normal metabolism activities of microalgal cells under N deprivation condition. Pyrroline-5-carboxylate reductase (PCR1; spot H48), which participates in arginine and proline metabolism, was dramatically increased by 18.34-fold. These generated amino acids might be used for protein synthesis to provide energy for normal metabolic activity in Chlorella cells under nutrient limitation conditions. A contradictory result exists between the protein synthesis herein and the inhibition of protein synthesis above. This inconsistency in results suggests that the regulatory mechanism of protein synthesis in cells after N deprivation is complicated. N deprivation promotes the activity of carbohydrate metabolism in C. protothecoides. Some up-regulated proteins associated with carbohydrate metabolism were significantly increased in this study. Transketolase (TK; spot H2), an enzyme of PPP in all organisms and the Calvin cycle of photosynthesis, was enhanced by 8.93-fold. During photosynthetic damage, TK participates in PPP, which involves a 2carbon fragment from D-xylulose-5-P to aldose erythrose-4phosphate, resulting in fructose 6-phosphate and glyceraldehyde-3-P. Ultimately, TK connects PPP to glycolysis (Figure 5). NAD-dependent epimerase/dehydratase (SNE4; spot H7), glucose-1-phosphate adenylyltransferase (STA6; spot H12), and two nucleotide sugar epimerases (SNE1, spot H26; SNE4, spot H30) also increased by 5.12-, 15.22-, and 3.20-fold, respectively. These compounds are related to starch metabolism. Spot H30 (SNE4) was a newly induced protein in cells, compared with the control group (BCM). In starch metabolism, glucose was first converted into UDPG or ADPG by SNE1 and SNE4, and then UDPG or ADPG participated in starch accumulation by STA6 (Figure 5). In the present study, the algal cells were grown in BCM medium (heterotrophism phase), utilizing glucose for cell growth and starch accumulation, which is the basic stored energy form of microalgae. When the late exponential algal cells were reinoculated into BCM-N (N deprivation phase), starch metabolized and penetrated into the glycolysis route (Figure 5), providing stock and intermediate of lipid and protein synthesis. Two proteins (FBA1, spot H31; FBA4, spot H40) were dramatically enhanced by 11.80- and 25.47-fold, respectively. These proteins also participate in glycolysis. As shown in Figure 5, the conversion of FB to glyceraldehyde-3phosphate and dihydroxyacetone phosphate is catalyzed by FB aldolase (spots H31and H40). These results suggest enhanced carbohydrate metabolism under N deprivation. Malate dehydrogenase (MDH1; spot 47) is a cytosolic malate

5. CONCLUSION The HND was first employed to culture C. protothecoides for microalgal lipid production. The advantage of HND over previous strategies is its good balance between biomass and lipid productivity by merging the positive aspects of heterotrophism and nutrient limitation. For example, in this study, favorable results in terms of productivities of resulting biomass (13.89 g/L) and lipid (10 g/L) were achieved by HND. The biomass and lipid yields were 2- to 2.6-fold and 3.7to 5.0-fold higher, respectively, than those reported in the literature, as indicated in the text with the same C. protothecoides strain. In summary, HND has great potential for large-scale cultivation of microalgae to produce alga-based biodiesel. Cell proteomic analysis was conducted to reveal the underlying molecular basis of lipid accumulation in response to HND mode. This study identified 33 differentially expressed proteins in C. protothecoides cells, including 15 down-regulated proteins involved in photosynthesis, protein synthesis and folding, gene regulation and β-oxidation of fatty acids; 13 upregulated proteins related to carbohydrate metabolism, stress response and defense, amino acid biosynthesis, and secondary metabolite biosynthesis; and 5 hypothetical proteins. CarbohyI

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(23) Kurian, D.; Phadwa, K.; Maenpaa, P. Proteomics 2006, 6, 3614− 3624. (24) Xu, N.; Zhang, X.; Fan, X.; Han, L.; Zeng, C. J. Appl. Phycol. 2001, 13, 463−469. (25) Li, Y.; Horsman, M.; Wu, N.; Lan, C. Q.; Dubois-Calero, N. Biotechnol. Prog. 2008, 24, 815−820. (26) Hu, Q.; Sommerfeld, M.; Jarvis, E.; Ghirardi, M.; Posewitz, M.; Seibert, M.; Darzins, A. Plant. J. 2008, 54, 621−639. (27) Deng, X.; Li, Y.; Fei, X. Afr. J. Microbiol. Res. 2009, 3, 1008− 1014. (28) Perez-Garcia, O.; Escalante, F. M.; de-Bashan, L. E.; Bashan, Y. Water. Res. 2011, 45, 11−36. (29) Zeng, X. H.; Danquah, M. K.; Chen, X. D.; Lu, Y. H. Renewable Sustainable Energy Rev. 2011, 15, 3252−3260. (30) Xu, Y. H.; Liu, R.; Yan, L.; Liu, Z. Q.; Jiang, S. C.; Shen, Y. Y.; Wang, X. F.; Zhang, D. P. J. Exp. Bot. 2012, 63, 1095−1106. (31) Li, Y. J.; Fei, X. W.; Deng, X. D. Biomass Bioenergy 2012, 42, 199−211. (32) Pandey, S.; Rai, R.; Rai, L. C. J. Proteomics 2012, 75, 921−937. (33) Derelle, E.; Ferraz, C.; Rombauts, S.; Rouzé, P.; Worden, A. Z.; Robbens, S.; Partensky, F.; Degroeve, S.; Echeynié, S.; Cooke, R.; Saeys, Y.; Wuyts, J.; Jabbari, K.; Bowler, C.; Panaud, O.; Piégu, B.; Ball, S. G.; Ral, J. P.; Bouget, F. Y.; Piganeau, G.; De Baets, B.; Picard, A.; Delseny, M.; Demaille, J.; Van de Peer, Y.; Moreau, H. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 11647−11652. (34) Molnar, A.; Bassett, A.; Thuenemann, E.; Schwach, F.; Karkare, S.; Ossowski, S.; Weigel, D.; Baulcombe, D. Plant. J. 2009, 58, 165− 174. (35) Contreras, L.; Moenne, A.; Gaillard, F.; Potin, P.; Correa, J. A. Aquat. Toxicol. 2010, 96, 85−89. (36) Grudkowska, M.; Zagdanska, B. Acta Biochim. Pol. 2004, 51, 609−624. (37) Zhang, Y.; Wolfe, D. M.; Pohlmann, E. L.; Conrad, M. C.; Roberts, G. P. Microbiology 2006, 152, 2075−2089. (38) Minárik, P.; Tomásková, N.; Kollárová, M.; Antalík, M. Gen. Physiol. Biophys. 2002, 21, 257−265. (39) Aliaga Goltsman, D. S.; Denef, V. J.; Singer, S. W.; VerBerkmoes, N. C.; Lefsrud, M.; Mueller, R. S.; Dick, G. J.; Sun, C. L.; Wheeler, K. E.; Zemla, A.; Baker, B. J.; Hauser, L.; Land, M.; Shah, M. B.; Thelen, M. P.; Hettich, R. L.; Banfield, J. F. Appl. Environ. Microbiol. 2009, 75, 4599−4615. (40) Dunn, M. F.; Ramirez-Trujill, J. A.; Hernandez-Lucas, I. Microbiology 2009, 155, 3166−3175.

drate metabolism and inhibition of fatty acid catabolism (βoxidation) achieved lipid accumulation in algal cells by the KEGG pathway. As shown in Figure 5, pyruvate and acetylCoA have important functions in the direction of carbon flow to lipid. In future studies, further functional analysis of these altered proteins would help elucidate the complicated relationship between cell growth and lipid accumulation in microalgae.

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AUTHOR INFORMATION

Corresponding Authors

*Phone: 86-731-58629863. Fax: 86-731-58298172. E-mail: [email protected]; [email protected] (Y. Li). *E-mail: [email protected] (B. Feng). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21105085 and 31270988), the China Postdoctoral Science Foundation funded project (2012M521531), and the Hunan Provincial Natural Science Foundation of China (13JJ3062).

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REFERENCES

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