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Abstract
Oleaginous microalgae have been emerging as the third-generation feedstocks for biofuel production. Genetic manipulation for improving triacylglycerol (TAG) accumulation represents a promising approach towards the economics of microalgal biofuels. Acetyl-CoA, the essential carbon precursor for de novo fatty acid biosynthesis, can be derived from pyruvate catalyzed by pyruvate dehydrogenase, which is negatively regulated by pyruvate dehydrogenase kinase (PDK). In the present study, we characterized a PDK gene (NsPDK) from Nannochloropsis salina. Subcellular localization assay assisted by green fluorescence protein (GFP) fusion indicated the localization of NsPDK in mitochondria of N. salina cells. NsPDK knockdown via RNA interference strategy attenuated NsPDK expression at the mRNA level and its enzymatic activity in vivo, leading to faster TAG accumulation without compromising cell growth under high light stress conditions. Interestingly, the TAG increase was accompanied by a decline in membrane polar lipids. NsPDK knockdown also altered fatty acid profile in N. salina. Furthermore, transcriptional analysis suggested that the carbon metabolic pathways might be influenced by NsPDK knockdown leading to diverted carbon flux towards TAG synthesis. Taken together, our results demonstrate the role of NsPDK in regulating TAG accumulation and provide valuable insights into future manipulation of oleaginous microalgae for improving biofuel production.
Highlights
The increasing concerns about the shortage of fossil fuels and the burning-associated environmental problems have led to interest in developing sustainable and renewable biofuels in recent years[1]
The phylogenetic analysis based on amino acid sequences revealed that the putative N. salina PDK sequence (NsPDK) was closely clustered with pyruvate dehydrogenase kinase (PDK) from N. gaditana and N. oceanica IMET1 (Fig. 1)
We further studied the effect of NsPDK knockdown on total fatty acid profile (Fig. 5d)
Summary
The increasing concerns about the shortage of fossil fuels and the burning-associated environmental problems have led to interest in developing sustainable and renewable biofuels in recent years[1]. Li et al.[9] found out that most of the genes involved in de novo FA synthesis were downregulated while total fatty acid accumulation was significantly enhanced in Nannochloropsis under nitrogen starvation This could explain why previous attempts to increase lipid content by overexpressing acetyl-coA carboxylase or fatty acid synthase had limited success[10,11,12]. NsPDK knockdown via RNA interference strategy promoted total lipid content and altered fatty acid profiles, and enhanced TAG accumulation, while without slowing down cell growth. Transcriptional analysis suggested the possible gene regulatory network of PDK and its knockdown may bring more carbon precursors and reducing powers for de novo fatty acid synthesis thereby enhancing TAG accumulation. Our work helps understand the role of PDK in regulating carbons for lipid biosynthesis and provides insights into target genetic engineering of Nannochloropsis for trait improvement and production uses
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