Genetic transformation of green alga Haematococcus pluvialis for the regulation of carotenoid biosynthesis

Haematococcus pluvialis is a unicellular green alga which produces a
ketocarotenoid, astaxanthin having pharmaceutical and nutraceutical applications owing
to its high antioxidant activity. Biotechnological approaches like genetic transformation
methods (Agrobacterium mediated), cloning strategies, etc are essential to
improve/regulate this ketocarotenoid in H. puvialis. For the standardization of
Agrobacterium mediated transformation procedures for algae, preliminary studies like,
sensitivity of algae for different antibiotics, media selection for the co-cultivation for both
algae and the bacteria were carried out. The sensitivity studies showed that the H.
pluvialis was able to tolerate up to the concentrations of 2000 mg L-1 of antibiotics,
cefotaxime and augmentin. Growth and multiplication of the algae was suppressed and
ultimately killed when hygromycin concentration exceeded 2 mg L-1 in solid media. No
growth was observed even after 4 weeks of inoculation when the hygromycin
concentration exceeds 2 mg L-1. Among the different cocultivation medium (BBM + half
strength of LB medium, Z8 medium + half strength of LB medium, Z8 medium + 0.5%
mannitol, Z8 medium only and Tris - acetate - phosphate (TAP)) medium were tested ,
only the TAP medium favoured the growth of both the alga and Agrobacterium. Colonies
resistant to hygromycin at 10 mgL-1 expressed β-glucuronidase (GUS) and green
fluorescence protein (GFP). PCR was used to successfully amplify fragments of the hpt
(407 bp) and GUS (515 bp) genes from transformed cells while southern blots indicated
the integration of hygromycin gene into the genome of H. pluvialis. Scanning electron
microscopy indicated that the cell wall of H. pluvialis was altered on infection with
Agrobacterium. The transformation achieved here by Agrobacterium does not need
treatment with acetosyringone or the wounding of cells. The carotenoid profile of the
transformed H. pluvialis showed no difference between the control cells.
The amplification of b-carotene ketolase (BKT) was observed for the different primers
synthesized. No amplification was observed for the any of the BKH primers studied. The
1.8 kb amplicon of BKT gene was cloned to a cloning vector pRT100 in between the
CaMV 35S promoter and the poly A region. The b-carotene ketolase gene from cloned
plasmid pRT100 was further transferred to a binary vector pCAMBIA1304. Sequence
analysis of cloned BKT for nucleotide and amino acid showed 99% similarity of the
reported BKT gene accession number D45881. Six exons and five introns were observed
for the BKT gene cloned from the H. pluvialis. Even though there were few nucleotide
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polymorphism, there is no shift in the reading frame. The cloned plasmid pRT100 and
pCAMBIA1304-BKT were further confirmed by restriction digestion. Standardized
Agrobacterium mediated genetic transformation protocol was followed to transform the
cloned binary vector having BKT (pCAMBIA1304-BKT) gene to the H. pluvialis.
Confirmation of the transformation of cloned BKT to the H. pluvialis was studied by
analyzing the GUS, GFP expression. PCR analysis for the CaMV 35S primers showed
2.5 kb of the BKT gene with the promoter and poly A region. For the BKT forward
primer and the CaMV 35S reverse primer also the exact size of amplicon was observed.
Southern blotting also showed the difference in the banding pattern of the enzyme
digested plasmid and the transformed H. pluvialis. No bands were observed for the
control cells. Transcript level analysis of the BKT showed 3 to 4 fold higher expression
of BKT in transformants than the control cells. The transformed cells were subjected to
different stress condition for the induction of secondary carotenoids. Total carotenoids
and astaxanthin content in transformed cells showed 2-3 folds higher when compared to
the control. Astaxanthin was found to be higher (7.96 mg/g) in sodium acetate (4.4 mM)
treated culture followed by sodium acetate 4.4mM and NaCl 0.25% (4.99 mg/g). The
intermediates like canthaxanthin and echinenone were the major carotenoids which are
present more in the transformants but not in the control H. pluvialis. It shows that
echinenone and canthaxanthin content were approximately 8 to 10 fold higher in sodium
acetate 4.4mM and NaCl 0.25% treated culture. The NaCl + sodium acetate treated
culture in transformed cells showed higher level of expression for all the enzymes
studied. It was observed that the expression levels of PSY, PDS, LCY, BKT and BKH
were 8.8, 5.8, 11.7, 8.1 and 6.4 fold higher respectively when compared to the control
green cells. But when compared to the transformed green cells it is 6.1, 1.7, 22.7, 4.0 and
2.9 fold higher respectively.
This first successful Agrobacterium mediated transformation in green micro alga
Haematococcus pluvialis would pave the way for manipulation of many important
pathways relevant to food, pharmaceutical and nutraceutical industries. Further the
cloned BKT gene from H. pluvialis will be used for higher production of carotenoids
through transformation in the heterologous host like Duanliella sp, Daucus sp,
Lycopersicum sp to regulate the carotenoid biosynthesis. Therefore this study helps in
production of higher level of carotenoids through the Agrobacterium mediated
transformation system in carotenoid producing organisms using the cloned BKT gene.