Transcriptome analysis of Haematococcus pluvialis of multiple defensive systems against nitrogen starvation

Highlights

• Transcriptome analysis was used to elucidate the multiple defensive systems against nitrogen starvation in H. pluvialis.

• Carbon fixation, glycolysis, fatty acid and carotenoid biosynthesis pathways were enhanced.

• C4 and C3 pathway coexisted and they were both stimulated to promote carbon fixation under nitrogen starvation.

• The isoprene unit, IPP was originated from MEP pathway.

• TAG was synthesized through both acyl-CoA dependent and independent pathway.

Abstract

Haematococcus pluvialis could accumulate large amounts of triacylglycerol (TAG) and astaxanthin under various environmental stresses. To gain insights into the multiple defensive systems for carbon metabolism against nitrogen starvation, transcriptome analysis was performed. It was found that the genes related to carbon fixation, glycolysis, fatty acid and carotenoid biosynthesis pathways were up-regulated remarkably. Glyceraldehyde 3-phosphate (G3P) biosynthesis was accelerated with the enhanced C3 and C4 pathway. Meanwhile, the pyruvate kinase (PK) and pyruvate dehydrogenase E2 component (aceF) genes were significantly increased 12.9-fold and 13.9-fold, respectively, resulting more pyruvate and acetyl-CoA generation, which were beneficial to carotenoids and fatty acid biosynthesis. Methylerythritol 4-phosphate (MEP) pathway mediated carotenoid precursor isopentenyl diphosphate (IPP) synthesis, as the all eight related genes were up-regulated. The carbon flux toward astaxanthin biosynthesis with the increased astaxanthin pathway genes. The redistribution of carbon was also promoted for TAG accumulation. In addition, the up-regulation of diacylglycerol acyltransferase (DGAT) and phospholipid: diacylglycerol acyltransferase (PDAT) genes indicated that both acyl-CoA dependent and independent pathway regulated TAG accumulation. Therefore, this work reveals the multiple defensive mechanism for carbon metabolism in response to nitrogen starvation, which extended our understanding on the carotenoids, TAG and other important metabolites synthesis.

Introduction

Microalgae with high productivity of areal biomass can accumulate a large quantity of high-value products, such as carotenoids and polyunsaturated fatty acids which are produced via primary metabolism or induced as a secondary metabolites in response to various stresses [[1], [2], [3]]. Haematococcus pluvialis is a green microalga and regarded as the best natural resource for its ability to synthetize and store large amounts of the carotenoid astaxanthin under stress conditions, including high light stress [4,5], nitrogen starvation [3,6], culture aging [7], high light combined with nitrogen or phosphorus starvation [8,9], nutrient stress [10], salinity [11], high temperature [12,13] and chemical modulators [14]. The carotenoid astaxanthin (3,3’-dihydroxy-β,β-carotene-4,4’-dione) is called “super vitamin E” due to its strong antioxidant properties and is a high-value red pigment widely applied in a range of fields, such as aquaculture, nutraceutical, pharmaceutical, and cosmetic industries [15,16].

Though some other microalgae, aquatic animals, and yeast can also produce astaxanthin, H. pluvialis is proved to be more efficient to accumulate astaxanthin and higher commercial profits [17,18]. The global market transaction of astaxanthin can reach over $250 million per year [19,20]. However, the astaxanthin content in H. pluvialis can usually only reach up to 4 % of cell dry weight [14]. In addition, lipid is assumed to be the substrate of astaxanthin esterification and storage for astaxanthin ester [6,9,21,22]. Therefore, further exploration and exploitation of the underlying carbon regulatory mechanism of the astaxanthin and lipid biosythiesis for improving the astaxanthin productivity in H. pluvialis have attracted significant attention in recent years [[23], [24], [25]].

The molecular mechanisms involved in astaxanthin accumulation through assaying the genes and their expressions related to astaxanthin biosynthesis in H. pluvialis have been studied by the global analytical approaches, such as proteomics [3,26,27], metabolomics and network analysis [8,28,29]. In the recent studies, transcriptome analysis is a powerful tool to identify gene expressions for the important metabolites biosynthesis and has been widely applied in microalgae to investigate the synthesis of astaxanthin, fatty acids and triacylglycerol [21,[30], [31], [32]]. In Chlorella zofingiensis, transcriptome analysis revealed that the expression levels of genes involving in astaxanthin biosynthesis were significantly up-regulated, coupled with the repression of side pathway genes on glucose condition [21]. For cold tolerance strain Chlamydomonas sp, transcriptome revealed a large number of genes related to the cold response, similar to other psychrophilic species [33]. Besides, the process has been employed to annotate the enzymes and transcription factors coding genes involving in lipid and carbon metabolism at logarithm and stationary growth phase [34], enrich the CA gene information in the database, demonstrate that the carbon fixation and partition are more active under high CO₂ level [35], proposed that chlorophyll a/b binding protein are associated with the utilization of inorganic carbon at low CO₂ condition [36].

Recent studies have showed that the response to nitrogen starvation leads to the accumulation of carbohydrates and fatty acids as well as increased activity of the tricarboxylic acid cycle in H. pluvialis. [6,10,35]. However, little is known about multiple defensive systems against nitrogen starvation for carbon metabolism, especially the genetic details and regulation mechanism leading to the biosynthesis of astaxanthin, fatty acids and triacylglycerol. Therefore, a genome-wide investigation, accompanied with differential gene expression analysis, would be of importance and necessity to identify all genes involving biosynthesis of astaxanthin, fatty acids and triacylglycerol biosynthesis in H. pluvialis. However, the genome sequence for quantifying the transcriptome was not accessible, led to slow investigation of global transcriptomic analysis [37,38].

To gain detailed genetic information for revealing the resistance mechanism, unigenes of H. pluvialis were assembled by transcriptomic data and then annotated by SwissProt, KEGG, and COG. We systematically examined differential gene expression responding to nitrogen starvation in order to identify genes involved in general response to the stress condition. Furthermore, we comprehensively compared differentially expressed genes involved in carbon fixation pathway for astaxanthin biosynthesis, lipid metabolism, based on the mode of action of nitrogen starvation. The assembled, annotated transcriptome sequences provide a valuable genomic resource for further understanding the molecular mechanism of Haematococeus pluvialis under nitrogen starvation. In addition, the results were supported by the qRT-PCR analysis, the changes of cell morphology and contents of intracellular pigments and lipid.