Photoprotective carbon redistribution in mixotrophic Haematococcus pluvialis under high light stress.

Mixotrophy of Haematococcus pluvialis is a potential strategy for producing astaxanthin. However, this strategy has not been extensively commercialized because the mixotrophic mechanisms by which H. pluvialis overcomes high light stress are unclear. This study analyzed the biochemical compositions and differential proteomics of mixotrophic H. pluvialis under different light conditions. High light exposure substantially increased astaxanthin, carbohydrate, and fatty acid contents. A total of 119 and 81 proteins were significantly up- and down-regulated after two days of high light exposure. These proteins mainly enriched pathways for photosynthetic metabolism, glyoxylate cycle, and biosynthesis of secondary metabolites. This study proposed a regulatory model through which mixotrophic H. pluvialis copes with high light stress. The model includes pathways for modulating photosynthetic apparatus, increasing astaxanthin accumulation by enhancing photorespiration, pentose phosphate and Embden-Meyerhof-Parna pathways, while thickening the cell wall by malate-oxaloacetate shuttle.

Haematococcus pluvialis Accumulated Lipid and Astaxanthin in a Moderate and Sustainable Way by the Self-Protection Mechanism of Salicylic Acid Under Sodium Acetate Stress.

To elucidate the mechanism underlying increased fatty acid and astaxanthin accumulation in Haematococcus pluvialis, transcriptome analysis was performed to gain insights into the multiple defensive systems elicited by salicylic acid combined with sodium acetate (SAHS) stresses with a time course. Totally, 112,886 unigenes and 61,323 non-repeat genes were identified, and genes involved in carbon metabolism, primary and secondary metabolism, and immune system responses were identified. The results revealed that SA and NaAC provide both energy and precursors to improve cell growth of H. pluvialis and enhance carbon assimilation, astaxanthin, and fatty acids production in this microalga with an effective mechanism. Interestingly, SA was considered to play an important role in lowering transcriptional activity of the fatty acid and astaxanthin biosynthesis genes through self-protection metabolism in H. pluvialis, leading to its adaption to HS stress and finally avoiding massive cell death. Moreover, positive correlations between 15 key genes involved in astaxanthin and fatty acid biosynthesis pathways were found, revealing cooperative relation between these pathways at the transcription level. These results not only enriched our knowledge of the astaxanthin accumulation mechanism in H. pluvialis but also provided a new view on increasing astaxanthin production in H. pluvialis by a moderate and sustainable way in the future.

Transcription Factors From Haematococcus pluvialis Involved in the Regulation of Astaxanthin Biosynthesis Under High Light-Sodium Acetate Stress.

The microalgae Haematococcus pluvialis attracts attention for its ability to accumulate astaxanthin up to its 4% dry weight under stress conditions, such as high light, salt stress, and nitrogen starvation. Previous researches indicated that the regulation of astaxanthin synthesis might happen at the transcriptional level. However, the transcription regulatory mechanism of astaxanthin synthesis is still unknown in H. pluvialis. Lacking studies on transcription factors (TFs) further hindered from discovering this mechanism. Hence, the transcriptome analysis of H. pluvialis under the high light-sodium acetate stress for 1.5 h was performed in this study, aiming to discover TFs and the regulation on astaxanthin synthesis. In total, 83,869 unigenes were obtained and annotated based on seven databases, including NR, NT, Kyoto Encyclopedia of Genes and Genomes Orthology, SwissProt, Pfam, Eukaryotic Orthologous Groups, and Gene Ontology. Moreover, 476 TFs belonging to 52 families were annotated by blasting against the PlantTFDB database. By comparing with the control group, 4,367 differentially expressed genes composing of 2,050 upregulated unigenes and 2,317 downregulated unigenes were identified. Most of them were involved in metabolic process, catalytic activity, single-organism process, single-organism cellular process, and single-organism metabolic process. Among them, 28 upregulated TFs and 41 downregulated TFs belonging to 27 TF families were found. The transcription analysis showed that TFs had different transcription modules responding to the high light and sodium acetate stress. Interestingly, six TFs belonging to MYB, MYB_related, NF-YC, Nin-like, and C3H families were found to be involved in the transcription regulation of 27 astaxanthin synthesis-related genes according to the regulatory network. Moreover, these TFs might affect astaxanthin synthesis by directly regulating CrtO, showing that CrtO was the hub gene in astaxanthin synthesis. The present study provided new insight into a global view of TFs and their correlations to astaxanthin synthesis in H. pluvialis.

Transcriptome-based analysis of the effects of salicylic acid and high light on lipid and astaxanthin accumulation in Haematococcus pluvialis.

BACKGROUND: The unicellular alga Haematococcus pluvialis has achieved considerable interests for its capacity to accumulate large amounts of triacylglycerol and astaxanthin under various environmental stresses. To our knowledge, studies focusing on transcriptome research of H. pluvialis under exogenous hormones together with physical stresses are rare. In the present study, the change patterns at transcriptome level were analyzed to distinguish the multiple defensive systems of astaxanthin and fatty acid metabolism against exogenous salicylic acid and high light (SAHL) stresses. RESULTS: Based on RNA-seq data, a total of 112,463 unigenes and 61,191 genes were annotated in six databases, including NR, KEGG, Swiss-Prot, PFAM, COG and GO. Analysis of differentially expressed genes (DEGs) in KEGG identified many transcripts that associated with the biosynthesis of primary and secondary metabolites, photosynthesis, and immune system responses. Furthermore, 705 unigenes predicted as putative transcription factors (TFs) were identified, and the most abundant TFs families were likely to be associated with the biosynthesis of astaxanthin and fatty acid in H. pluvialis upon exposure to SAHL stresses. Additionally, majority of the fifteen key genes involved in astaxanthin and fatty acid biosynthesis pathways presented the same expression pattern, resulting in increased accumulation of astaxanthin and fatty acids in single celled H. pluvialis, in which astaxanthin content increased from 0.56 ± 0.05 mg·L-1 at stage Control to 0.89 ± 0.12 mg·L-1 at stage SAHL_48. And positive correlations were observed among these studied genes by Pearson Correlation (PC) analysis, indicating the coordination between astaxanthin and fatty acid biosynthesis. In addition, protein-protein interaction (PPI) network analysis also demonstrated that this coordination might be at transcriptional level. CONCLUSION: The results in this study provided valuable information to illustrate the molecular mechanisms of coordinate relations between astaxanthin and fatty acid biosynthesis. And salicylic acid might play a role in self-protection processes of cells, helping adaption of H. pluvialis to high light stress.

A novel fed-batch strategy to boost cyst cells production based on the understanding of intracellular carbon and nitrogen metabolism in Haematococcus pluvialis.

Haematococcus pluvialis is a prominent feedstock of astaxanthin. The ratio of carbon to nitrogen (C/N) strongly influences the metabolic pathways of mixotrophic-grown microalgae, however, its role involved in astaxanthin biosynthesis is still not fully understood. In this study, integrative metabolic and physiologic profiles were analyzed in elucidating how C/N affected carbon and nitrogen assimilation and thereby exerted influence on astaxanthin biosynthesis. It was demonstrated that high C/N up-regulated the activities of acetate kinase by increase of 5.76 folds in early logarithmic phase, leading a significant increase of acetyl-CoA. The increased carbon skeletons were then funneled into astaxanthin biosynthesis. Additionally, high C/N increased the proportion of carotenoid-intermediates in cytoplasm from chloroplast. Finally, a fed-batch cultivation strategy based on C/N gradient was developed. Biomass attained 9.18 g L-1 in 100% type of immotile cyst cells, which presented astaxanthin productivity at 15.45 mg L-1 d-1 afterward, exhibiting a promising paradigm in commercial production.

From Golden Rice to aSTARice: Bioengineering Astaxanthin Biosynthesis in Rice Endosperm.

Carotenoids are important phytonutrients with antioxidant properties, and are widely used in foods and feedstuffs as supplements. Astaxanthin, a red-colored ketocarotenoid, has strong antioxidant activity and thus can benefit human health. However, astaxanthin is not produced in most higher plants. Here we report the bioengineering of astaxanthin biosynthesis in rice endosperm by introducing four synthetic genes, sZmPSY1, sPaCrtI, sCrBKT, and sHpBHY, which encode the enzymes phytoene synthase, phytoene desaturase, ß-carotene ketolase, and ß-carotene hydroxylase, respectively. Transgneic overexpression of two (sZmPSY1 and sPaCrtI), three (sZmPSY1, sPaCrtI and sCrBKT), and all these four genes driven by rice endosperm-specific promoters established the carotenoid/ketocarotenoid/astaxanthin biosynthetic pathways in the endosperm and thus resulted in various types of germplasm, from the yellow-grained ß-carotene-enriched Golden Rice to orange-red-grained Canthaxanthin Rice and Astaxanthin Rice, respectively. Grains of Astaxanthin Rice were enriched with astaxanthin in the endosperm and had higher antioxidant activity. These results proved that introduction of a minimal set of four transgenes enables de novo biosynthesis of astaxanthin in the rice endosperm. This work provides a successful example for synthetic biology in plants and biofortification in crops; the biofortified rice products generated by this study could be consumed as health-promoting foods and processed to produce dietary supplements.

Metabolic engineering a yeast to produce astaxanthin.

In this study, an astaxanthin-biosynthesis Kluyveromyces marxianus strain Sm23 was first constructed, which could produce 31µg/g DCW astaxanthin. Then, repeated genome integration of the key astaxanthin biosynthesis genes Hpchyb and bkt was done to increase gene copy number and astaxanthin yield. Four improved strains were obtained and the yield of astaxanthin and the total yield of carotenoids in a strain increased with the copy numbers of Hpchyb and bkt. To improve the yield further, the gene Hpchyb from Haematococcus pluvialis was modified by site-directed mutagenesis to increase the enzyme efficiency or/and to prevent the heterologous protein degradation by ubiquitination. Using repeated-integration approach of bkt and the mutated Hpchyb into Sm23, the S3-2 strain was obtained and shown to produce the 3S, 3'S-astaxanthin at 9972µg/g DCW in a 5L fermentor.

Multiple transformation with the crtYB gene of the limiting enzyme increased carotenoid synthesis and generated novel derivatives in Xanthophyllomyces dendrorhous.

Xanthophyllomces dendrorhous (in asexual state named as Phaffia rhodozyma) is a fungus which produces astaxanthin, a high value carotenoid used in aquafarming. Genetic pathway engineering is one of several steps to increase the astaxanthin yield. The limiting enzyme of the carotenoid pathway is phytoene synthase. Integration plasmids were constructed for transformation with up to three copies of the crtYB gene. Upon stepwise transformation, the copy numbers of crtYB was continuously increased leading to an almost saturated level of phytoene synthase as indicated by total carotenoid content. Several carotenoid intermediates accumulated which were absent in the wild type. Some of them are substrates and intermediates of astaxanthin synthase. They could be further converted into astaxanthin by additional transformation with the astaxanthin synthase gene. However, three intermediates exhibited an unusual optical absorbance spectrum not found before. These novel keto carotenoid were identified by HPLC co-chromatography with reference compounds generated in Escherichia coli and one of them 3-HO-4-keto-7',8'-dihydro-ß-carotene additionally by NMR spectroscopy. The others were 4-keto-ß-zeacarotene and 4-keto-7',8'-dihydro-ß-carotene. A biosynthesis pathway with their origin from neurosporene and the reason for their synthesis especially in our transformants has been discussed.

Genomic organization of the structural genes controlling the astaxanthin biosynthesis pathway of Xanthophyllomyces dendrorhous

The cloning and nucleotide sequence of the genes (idi, crtE, crtYB, crtl and crtS) controlling the astaxanthin biosynthesis pathway of the wild-type ATCC 24230 strain of Xanthophyllomyces dendrorhous in their genomic and cDNA version were obtained. The idi, crtE, crtYB, crtl and crtS genes were cloned, as fragments of 10.9, 11.5, 15.8, 5.9 and 4 kb respectively. The nucleotide sequence data analysis indicates that the idi, crtE, crtYB, crtl and crtS genes have 4, 8,4, 11, and 17 introns and 5, 9, 5, 12 and 18 exons respectively. In addition, a highly efficient site-directed mutagenesis system was developed by transformation by integration, followed by mitotic recombination (the double recombinant method). Heterozygote idi (idi+ / idi-::hph), crtE (crtE+ / crtE -::hph), crtYB (crtYB + / crtYB -::hph), crtI (crtI+ / crtI-::hph) and crtS (crtS +/crtS -::hph) and homozygote mutants crtYB (crtYB -::hph/crtYB -::hph), crtI (crtI -::hph/crtI -::hph) and crtS (crtS -::hph / crtS -::hph) were constructed. All the heterozygote mutants have a pale phenotype and produce less carotenoids than the wild-type strain. The genetic analysis of the crtYB, crtl and crtS loci in the wild-type, heterozygote, and homozygote give evidence of the diploid constitution of ATCC 24230 strains. In addition, the cloning of a truncated form of the crtYB that lacks 153 amino acids of the N-terminal region derived from alternatively spliced mRNA was obtained. Their heterologous expression in Escherichia coli carrying the carotenogenic cluster of Erwinia uredovora result in trans-complementation and give evidence of its functionality in this bacterium, maintaining its phytoene synthase activity but not the lycopene cyclase activity.