Glycerol and neutral lipid production in the oleaginous marine diatom Phaeodactylum tricornutum promoted by overexpression of glycerol-3-phosphate dehydrogenase

Abstract

Background: Microalgae are ideal raw materials for biodiesel and bioactive compounds. Glycerol-3-phosphate is formed from dihydroxyacetone phosphate (DHAP) through the glycolytic pathway catalyzed by glycerol-3-phosphate dehydrogenase (GPDH). Results: GPDH was characterized in the marine diatom Phaeodactylum tricornutum. In the GPDH-overexpressing P. tricornutum cells, the glycerol concentration per cell in the transformed diatom increased by 6.8-fold compared with the wild type, indicating that the overexpression of GPDH promoted the conversion of DHAP to glycerol-3-phosphate. There was a 60% increase in neutral lipid content, reaching 39.7% of dry cell weight in transgenic cells in the stationary phase, despite a 20% decrease in cell concentration. Fatty acid profiling showed that the levels of 16-and 18-carbon monounsaturated fatty acids significantly increased. Conclusion: GPDH had a significant impact on numerous metabolic processes in diatom cells, including the biosynthesis of glycerol and neutral lipids. These findings are instructive for the metabolic engineering of microalgae for biofuel production.

Two Glycerol-3-Phosphate Dehydrogenases from Chlamydomonas Have Distinct Roles in Lipid Metabolism

“…Thus, if the increased abundance of GPD3 enzyme in a GPD3-OE line is further enhancing flux toward glycerolipid metabolism, as indicated by increased PA synthesis, then glycerol synthesis, in turn, may decline. This response is also distinct from the overexpression of GPDH in P. tricornutum, where glycerol content increased alongside a lipid increase (Yao et al, 2014). In D. tertiolecta, one GPDH isoform functions as an osmoregulator by producing glycerol, while the other is a glyceride form, producing TAG (Gee et al, 1993).…”

“…Some successful approaches have involved the inhibition of lipid catabolism pathways, bypass of carbohydrate synthesis, or modification of transcription factors (Trentacoste et al, 2013;Daboussi et al, 2014;Ngan et al, 2015), while other approaches, such as the overexpression of specific Kennedy pathway components like a DGAT gene, have been less successful in some instances (La Russa et al, 2012). Previous expression of a yeast GPDH in oilseed rape (Vigeolas et al, 2007) and the recent overexpression of a diatom GPDH (Yao et al, 2014) indicate the potential of GPDH manipulation. While the knockdown analysis of GPD2 and GPD3 identified these as potential candidate gene targets to achieve enhanced oil accumulation in microalgae, the C. reinhardtii GPDH overexpression phenotypes were not as expected and were intriguingly different from the P. tricornutum and oilseed rape examples.…”

“…Transcriptomic analyses have demonstrated that some of the C. reinhardtii GPDH genes, such as GPD2, GPD3, and GPD4, are induced under conditions that correlate with lipid induction (Goodenough et al, 2014). Overexpression of a GPDH gene in the diatom Phaeodactylum tricornutum gave increased glycerol alongside a subtle increase in total lipid content, which included higher monounsaturated fatty acids but lower polyunsaturated fatty acids compared with the wild type (Yao et al, 2014). There is also compelling evidence for an important role for GPDH in lipid metabolism from nonalgal studies.…”

The dilemma for lipid productivity in green microalgae: importance of substrate provision in improving oil yield without sacrificing growth

“…Channeling of carbon away from either of these pathways to TAG synthesis would reduce the carbon available for pathways supporting cellular growth, such as the tricarboxylic acid (TCA) cycle. Scientists have overexpressed G3PDH in the diatom Phaeodactylum tricornutum leading to a 60% increase in neutral lipids during stationary phase, as a 6.8-fold increase in glycerol content provided the backbone for TAG synthesis [61]. However, it also resulted in a 20% decrease in cell growth, as overexpressing G3PDH promoted the conversion of DHAP to G3P, shunting carbon away from glycolysis and the TCA cycle to form glycerol.…”

Knockdown of phosphoenolpyruvate carboxykinase increases carbon flux to lipid synthesis in Phaeodactylum tricornutum

“…However, many engineering efforts focused on increasing lipid biosynthesis or blocking the competing pathways of carbohydrate formation, while successfully increasing lipid content have resulted in concomitant growth decreases in engineered strains. For example, knockdown of the genes encoding pyruvate dehydrogenase kinase [28], and overexpression of type 2 diacylglycerol acyltransferase [33], glycerol-3-phosphate dehydrogenase [49], and NADP + -malic enzyme (ME2) [47] led to increased TAG accumulation in Phaeodactylum tricornutum, but the growth of these mutants were decreased to different extents. In addition, overexpression of acetyl CoA carboxylase did not increase oil production in the diatoms Cyclotella cryptica and Navicula saprophila [12].…”