Abstract
Constantly rising energy demands, finite fossil fuel reserves and
deteriorating environmental conditions have invoked worldwide interest for
exploring the sustainable sources of renewable biofuels. Locally adapted
photosynthetic oleaginous microalgae by tweaking with over expression of
transgenes responsible for higher lipid production could be an ideal way
producing the sustainable feedstock for biodiesel. Higher lipid biosynthesis is
an important aspect for an economic viability of biofuel production using
microalgae. To enhance lipid content, Chlorella
sorokiniana-I was genetically engineered with a key enzyme diacylglycerol
acyltransferase (BnDGAT2) from Brassica napus, responsible for neutral
lipid biosynthesis. The transformed colonies expressing aph7 gene, were selected on hygromycin-supplemented medium after
bombardment of algal cells using gene-gun. Higher rate of transformation
frequency was observed and transgene integration and expression were confirmed
by PCR, Southern blots, staining lipid droplets, proteins and
spectrofluorometric analysis of Nile red-stained cells. The physiological
parameters of transgenic C. sorokiniana
like growth, lipid and FAME content were comparatively studied with non-transformed
wildtype algal strain. The total lipid and fatty
acid methyl esters (FAME) were observed 1.5 and 3-fold respectively higher in
transgenic alga in comparison to wildtype. The heterogenous BnDGAT2 expression in C. sorokiniana has completely altered
the lipid composition. The ?-linolenic acid, an essential omega-3 fatty acid
(PUFA) was observed 50% higher in transgenic cells. The neutral lipid is a
major class (over 80% of total lipids) and most significant requirement for
biodiesel production; was remarkably doubled in the transformed alga than wildtype control.
The overexpression of BnDGAT2
significantly altered the fatty acids profile in the transformed alga. Results
of this study offer a valuable strategy of genetic manipulation for enhancing
polyunsaturated fatty acids and neutral lipids for biofuel production in algae.