Molecular and biochemical studies of astaxanthin biosynthesis in Haematococcus pluvialis

Haematococcus pluvialis is a commercially promising source of astaxanthin (3,3'- dihydroxy-β,β-carotene-4,4'-dione) which is mainly used as a pigmentation source in the aquaculture and poultry industries. The present work is focused on molecular and biochemical studies of astaxanthin biosynthesis in H. pluvialis. Nutrient stress (modified autotrophic medium containing 1/10th of N and P) and higher light intensity (60 μmol m-2 s-1) in combination with 17.1 mM NaCl / 4.4 mM sodium acetate enhanced total carotenoid and total astaxanthin content to 32.0 and 24.5 mg g–1 of dry biomass, respectively. Expression of carotenoid biosynthetic genes revealed that they are up-regulated and maximum transcript levels of phytoene synthase, phytoene desaturase, lycopene cyclase, -carotene ketolase and -carotene hydroxylase genes were found to be 158–277, 5–9, 470–674, 28–40, and 451–673-fold higher, respectively, than that in green vegetative cells under stress conditions. The maximum content of astaxanthin recorded in cells grown in medium with sodium acetate and NaCl/sodium acetate correlated with the expression profile of the astaxanthin biosynthetic genes. Both general carotenogenesis and secondary carotenoid induction were regulated at transcriptional and cytoplasmic translational levels. This study also suggested a possible involvement of acetate in the posttranscriptional modifications of carotenoid genes. Experiments using inhibitors of carotenoid and fatty acid synthesis indicated the involvement of other regulatory factors besides transcriptional regulation of carotenogenesis in H. pluvialis. Studies using photosynthetic inhibitors showed that the expression of photosynthetic genes, cabL1818, lhcbm9, psaB and rbcL were under redox control of plastoquinone pool and atpB gene expression may be regulated at cytochrome b6/f complex. The nuclear genes cabL1818 and lhcbm9 are coding for the chlorophyll a/b binding protein L1818 and major light-harvesting complex II m9 protein respectively. The chloroplast encoded psaB gene codes for PSI reaction centre protein PsaB. The chloroplast genes rbcL and atpB are coding for large subunit of Ribulose bisphosphate carboxylase oxygenase (Rubisco) and ATP synthase -subunit respectively. Expression of all five photosynthetic genes studied was regulated at transcriptional and cytoplasmic translational levels, and their expressions were reduced by norflurazon induced photo oxidative stress. Acetate modulates the high light induced expression of photosynthetic genes and it depends on redox state of cytochrome b6/f complex and cytoplasmic protein synthesis. The genes differentially expressed under the stress conditions were analysed by mRNA differential display RT PCR and 34 differentially expressed transcripts have been identified. These transcripts are having homology to molecules related to general metabolism, photosynthesis, carotenoid synthesis, lipid synthesis, tetrapyrrole synthesis, transporter proteins, defense signaling, genetic information processing and unknown function or shared no apparent homology to any expressed sequences in the GenBank/EMBL databases. A partial transcript homologue to psaB gene coding D1 protein of photosystem II has been identified for the first time in H. pluvialis. LCY and BKT activities were found to increase under stress condition. Maximum activity of BKT was observed in lipid globules of stress induced cultures. Pigment composition of cell fractions revealed that chloroplast fraction is having lutein, β- carotene and chlorophyll as pigments while lipid fraction is having β-carotene, astaxanthin, canthaxanthin and echinenone as pigments. Under the influence of HL, exposure of cells to nutrient deficiency enhanced carotenoid accumulation which was further enhanced by exposure to CO2 enriched environment and/or NaCl and sodium acetate addition. Changing the carbonate buffer in the lower compartment of 2-tier vessel to provide constant CO2 environment was found to be efficient in enhancing carotenoid content. Influence of phytohormones such as salicylic acid and methyl jasmonate on pigment production and antioxidant revealed that at lower concentrations these phytohormones could be used for elicitation of secondary carotenoid production. Influence of few cultural parameters and temperature treatments on regeneration efficiency of red cysts along with changes in pigment profile and expression of carotenogenic genes during regeneration were investigated. Regeneration efficiency has been improved by incubating less aged cyst cells in medium containing ammonium carbonate, 16:8 light dark cycles with light intensity of 30 μmol m-2 s-1. During regeneration there was decrease in total astaxanthin, total carotenoids and carotenoid to chlorophyll ratio, and increase in -carotene, lutein, total chlorophyll and chlorophyll a to b ratio. Expression analysis of carotenogenic genes during regeneration of H. pluvialis cysts showed these transcripts were transiently up-regulated upon transfer to favorable conditions and later reached basal expression levels of green motile vegetative cells. In addition, this is the first report of detection of carotenogenic gene transcripts in red cysts and their differential expression during regeneration. It is evident from the results that acetate has a role in regulation of both carotenogenic and photosynthetic gene expression. The results from the present studies will be helpful in understanding the regulation of carotenogenesis and metabolic engineering of carotenoid pathway.