Genetic transformation of Dunaliella bardawil with astaxanthin biosynthetic gene(s) from Haematococcus pluvialis.

Dunaliella bardawil is a unicellular marine microalga which is well known for its
halotolerant nature and for producing highest amount of β-carotene. However a stable
transformation methodology is lacking in this alga which is the major hurdle for its
genetic improvement and regulation of carotenoid pathway through metabolic
engineering.
For the standardisation of Agrobacterium mediated transformation in
D. bardawil, 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 various selection antibiotics tested against D. bardawil proved to be insensitive
even in higher concentrations in growth medium. The sensitivity studies showed that
the D. bardawil was able to tolerate cefotaxime and potassium clavulanate upto 1000
mgL-1 and 1500 mgL-1of antibiotics while minimum concentration of these antibiotics
that arrested growth of A. tumefaciens was determined to be 500 mgL-1 cefotaxime
and 300 mgL-1 potassium clavulanate respectively. Co-cultivation and selection was
carried out in solid TAP medium containing 0.2 M NaCl. The selection antibiotic
hygromycin was found to be effective in completely killing D. bardawil cells at a
concentration of 100 mgL-1. Hygromycin sensitivity of D. bardawil was found to be
inversely proportional to the NaCl concentration in the medium. The transformation
frequency observed with the addition of 100 μM acetosyringone was found to be
42.0 ± 3 per 106 cells plated. Addition of acetosyringone during co cultivation has no
effect on transformation frequency. Transformation was confirmed by GUS, GFP
expression, PCR for hpt gene, southern and western blotting. The site of integration of
trans-gene in the transformants was anlaysed by adaptor ligated genome walking
method and the sequences showed that integration has occured in non-coding regions
in the genomic DNA of transformants. Pigment and growth profile of transformants
were similar to that of wild D. bardawil. The transformants obtained through the
standardised protocol were found to be stable even in the absence of selection
antibiotic in the growth medium for a period of nearly 4 years. Molecular
characterisation of Dunaliella spp used in the present study using 18S rDNA showed
that the alga belongs to the category Dunaliella salina var teod, a hyper β- carotene
accumulating strain of Dunaliella salina.
viii
Inorder to increase the hygromycin sensitivity, glucose which is a known
inducer of H+-ATPase was added at 10 mM in the selection medium. A decrease in
hygromycin tolerance level from 100 to 25 mgL-1 was noted for HRC. Control cells
were sensitive to hygromycin levels as low as 12 mgL-1. H+-ATPase activation by
glucose was confirmed by medium acidification and H+-ATPase assay. The H+-
ATPase assay values indicated maximum activity of 18 μmol Pi-1 hour-1 mg-1 protein
at 10 mM concentration of glucose. Vanadate at 10 μM and DES at 15 μM
completely inhibited the activation by glucose which confirmed that H+-ATPase is
involved in medium acidification and that it is activated by glucose. There was slight
increase in the transformation frequency of treatments with 10 mM glucose. The
transformants were confirmed with PCR amplification of hpt from genomic DNA.
Identification of the phenolic compounds present in spent medium of D. bardawil
which may induce vir gene so as to facilitate gene transfer without acetosyringone
was carried out using HPLC and HPTLC methods. Major phenolics identified from
extracts from spent medium at basic pH were acetosyringone, vanillic acid, vanillin
and proto-catechuic acid out of which acetosyringone was observed as the major
phenolic compound. The compounds were confirmed with mass spectrometry (ESI
negative mode). The identified phenolic compounds were checked for their
transforming efficiency by incorporating them at 100 μM concentration in the cocultivation
medium. A maximum cell count of 95±8 per 106 cells was obtained by
addition of vanillin in the co-cultivation medium.
A binary vector harbouring bkt from H. pluvialis which is driven by Rubisco
smaller subunit promoter with its transit peptide was constructed. The genomic clone
of bkt2 was used in vector construction. The RBCS2 promoter region along with the
transit peptide was amplified from D. bardawil using adaptor ligated genomic walking
method. The 1.4 kb amplicon was confirmed by sequencing and was shown to have
99% similarity with other Rubisco sequences. The promoter obtained was analysed for
the presence of transcription factors and was shown to have several light responsive
elements. The presence of chloroplast targeted signal peptide was also analysed. The
amplicon was subsequently used for constructing two binary vector constructs; p1304-
RBB-BKT with 1.4 kb and p1304-RBS-BKT with 0.6 kb promoter regions.
Agrobacterium mediated transformation of D. bardawil was carried out using
ix
the standardised procedure using 4 binary vector constructs viz; p1304, p1304-CaMVBKT,
p1304-RBB-BKT and p1304-RBS-BKT.The selected transformants were
confirmed using GFP & GUS expression, PCR and southern blot anlaysis. The
expression of various carotenogenic genes PSY, PDS, LCY, BKT and CHY were
carried out in MAS100, De Walnes and MAS100 with ¼ strength phosphate and
nitrate media. The expression of BKT and CHY was higher in transformants with
RBCS promoter contructs, particularly with the deletion construct (p1304-RBS-BKT).
There is an upregulation in the endogenous hydroxylase level of transformants where
the BKT expression was higher. The expression of all genes was higher in nutrient
limiting medium. The analysis of carotenoids extracted from wild and transformant
Dunaliella were carried out using spectrophotometry, TLC, HPLC and MS (APCI).
Astaxanthin and canthaxanthin were detectable in p1304-RBB-BKT and p1304-RBSBKT
transformants in HPLC and MS analysis. There was a decrease in β-carotene and
lutein content in p1304-RBB-BKT and p1304-RBS-BKT. Maximum content of
astaxanthin observed in p1304-RBS-BKT transformant is 3.5 μgg-1 DW. The cDNA
clone of bkt was expressed in E. coli which showed an additional protein band of 33
KDa size in SDS-PAGE analysis. The presence of 33 KDa band was also observed in
p1304-CaMV-BKT, p1304-RBB-BKT and p1304-RBS-BKT transformants.
The full potential of a stable genetic transformation has not been realized
for Dunaliella, which is a commercially important microalga valued for its suitability
as a perfect candidate for molecular farming. Agrobacterium mediated genetic
transformation method described in the present study is a highly efficient
technique for developing stable transformants of Dunaliella which is most
recommended for production in outdoor conditions due to its halotolerant nature. The
standardised transformation methodology may be used for production of foreign
proteins in Dunaliella spp. which are relevant to food, pharmaceutical and
nutraceutical industries. The present study is the first report in genetic manipulation of
carotenoid biosynthetic pathway in D. bardawil for the production of keto-carotenoids
including astaxanthin. This robust transformation method would thus pave the way
for utilisation of D. bardawil for the production of valuable carotenoids through
metabolic engineering of carotenoid biosynthetic pathway.