Engineering Algae for Enhanced Lipid Accumulation in Biodiesel Production
DOI:
https://doi.org/10.5281/zenodo.22688099Keywords:
Nannochloropsis oceanica, diacylglycerol acyltransferase, CRISPR-Cas9, triacylglycerol, biodiesel, microalgal lipid engineeringAbstract
Microalgal triacylglycerol (TAG) is widely regarded as one of the more realistic feedstocks for renewable diesel,
yet wild-type strains rarely accumulate enough lipid, quickly enough, to make the economics work. We used
CRISPR-Cas9 to overexpress a type-2 diacylglycerol acyltransferase (DGAT2A) under its native
nitrogen-responsive promoter in the marine eustigmatophyte Nannochloropsis oceanica, and separately used
CRISPR interference to knock down ADP-glucose pyrophosphorylase (AGPase), the committed step of competing
chrysolaminarin storage carbohydrate synthesis, generating single and stacked engineered lines alongside
wild-type and empty-vector controls. Under nitrogen-replete growth the four genotypes were indistinguishable in
biomass productivity, but seven days into nitrogen starvation the stacked DGAT2A-OE / AGPase-KD line reached
54.8% lipid content by dry weight, versus 31.2% in wild type, without a corresponding drop in areal biomass
productivity that would have erased the gain on a per-litre basis. Gas chromatography-mass spectrometry of
derivatized fatty acid methyl esters (FAMEs) showed the engineered lines retained a biodiesel-relevant fatty acid
profile dominated by palmitic (16:0) and oleic (18:1) acids, with a modest increase in the saturation index that is
favourable for oxidative stability and cetane number. A twelve-day outdoor raceway-pond trial reproduced the
laboratory lipid advantage at roughly two-thirds of the indoor magnitude, consistent with the lower and more
variable irradiance and higher culture temperature the ponds experienced relative to the photobioreactor. These
results indicate that redirecting carbon flux away from chrysolaminarin and into the acyl-CoA-dependent TAG
synthesis route, rather than either intervention alone, is an effective and outdoor-transferable strategy for raising
volumetric lipid productivity in an industrially relevant Nannochloropsis background.
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