Transcriptome analysis reveals unique C4-like photosynthesis and oil body formation in an arachidonic acid-rich microalga Myrmecia incisa Reisigl H4301
Long-Ling Ouyang
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Si-Hong Chen
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Yan Li
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Zhi-Gang Zhou
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College of Aqua-life Sciences and Technology, Shanghai Ocean University
,
999 Hucheng Huan Road, Pudong New District, Shanghai 201306
,
China
Background: Arachidonic acid (ArA) is important for human health because it is one of the major components of mammalian brain membrane phospholipids. The interest in ArA inspired the search for a new sustainable source, and the green microalga Myrmecia incisa Reisigl H4301 has been found a potential ArA-producer due to a high content of intracellular ArA. To gain more molecular information about metabolism pathways, including the biosynthesis of ArA in the non-model microalga, a transcriptomic analysis was performed. Results: The 454 pyrosequencing generated 371,740 high-quality reads, which were assembled into 51,908 unique sequences consisting of 22,749 contigs and 29,159 singletons. A total of 11,873 unique sequences were annotated through BLAST analysis, and 3,733 were assigned to Gene Ontology (GO) categories. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis uncovered a C4-like photosynthesis pathway in M. incisa. The biosynthesis pathways of lipid particularly those of ArA and triacylglycerol (TAG) were analyzed in detail, and TAG was proposed to be accumulated in oil bodies in the cytosol with the help of caleosin or oil globule-associated proteins. In addition, the carotenoid biosynthesis pathways are discussed. Conclusion: This transcriptomic analysis of M. incisa enabled a global understanding of mechanisms involved in photosynthesis, de novo biosynthesis of ArA, metabolism of carotenoids, and accumulation of TAG in M. incisa. These findings provided a molecular basis for the research and possibly economic exploitation of this ArA-rich microalga.
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Background
Arachidonic acid (ArA, 20:45, 8, 11, 14), an -6 long-chain
polyunsaturated fatty acid (PUFA), is one of the major
components of mammalian brain membrane
phospholipids. It makes up approximately 20% of the total fatty
acids along with docosahexaenoic acid (22:63, DHA) in
the brain [1]. In addition, ArA is an important precursor
for the biosynthesis of diverse eicosanoids, such as
prostaglandins, leukotrienes and thromboxanes, which play
important roles in smooth muscle stimulation, platelet
aggregation control and the release of histamine during
anaphylactic shock and other allergic reactions [2]. ArA
deficiencies have been associated with neuro-visual
development disorders and other premature birth
complications [3-6]. ArA is therefore considered an essential
nutrient in addition to DHA during early infant
development and is suggested to be added into baby formula by
the Food and Agriculture Organization (FAO). Besides,
carotenoids are essential nutrients and important health
beneficial compounds with respect to their antioxidant
properties and ability to alleviate chronic diseases [7].
Human beings are mostly incapable to synthesize
carotenoids de novo, therefore, they rely upon diet to
obtain these compounds.
ArA can be commercially obtained from marine fish
oil, animal tissues and fungi [8]. Interest in ArA and
other long-chain PUFAs inspired the search for new
PUFA sources. Myrmecia incisa Reisigl H4301, a coccoid
green microalga species of Trebouxiophyceae [9], has
recently been reported to accumulate an unprecedentedly
high amount of ArA-rich triacylglycerols (TAG) in
cytoplasmic lipid bodies [10]. When M. incisa was cultured
under nitrogen starvation for 27 d, its ArA content
increased from 1.9% to 7.0% of dry weight (DW)
biomass and 76% of the intracellular ArA accumulated in
the form of neutral lipids [10]. In addition, carotenoids
content increased as well under nitrogen starvation in
Parietochloris incisa (synonym with M. incisa [11,12])
[13]. Thus, M. incisa may be a potential resource for
both ArA and carotenoids exploitation.
There are two pathways proposed in microalgae for
ArA biosynthesis [14-16]. The understanding of ArA
biosynthesis pathway in M. incisa, thus, is first of all for
the improvement of ArA level. Therefore, genes
encoding fatty acid desaturases and elongase of this microalga
have been characterized, and some routes were
determined by heterologous expression of these genes in
model organisms [10,17-19]. Carotenogenesis pathways
in microalgae are similar to those in higher plants on
one hand, and possess microalgae-specific features on
the other hand [20]. Some common carotenogenesis
genes have been identified in microalgae [20-22],
whereas the molecular information for carotenoids
biosynthesis in M. incisa is rather limited [10,11].
Expressed sequence tag (EST) analysis is the primary
tool for novel gene discovery, particularly in non-model
organisms for which full genome sequencing is not
economically feasible. By using Sanger sequencing, a cDNA
library from M. incisa generated only 595 unique
sequences from 1,854 readable ESTs with little information
of interest [11]. In contrast to this (...truncated)