Oleaginous photosynthetic organisms such as microalgae are promising sources for biofuel production through the generation of carbon-neutral sustainable energy. However, the metabolic mechanisms driving high-rate lipid production in these oleaginous organisms remain unclear, thus impeding efforts to improve productivity through genetic modifications. We analyzed the genome and transcriptome of the oleaginous diatom Fistulifera solaris JPCC DA0580. Next-generation sequencing technology provided evidence of an allodiploid genome structure, suggesting unorthodox molecular evolutionary and genetic regulatory systems for reinforcing metabolic efficiencies. Although major metabolic pathways were shared with nonoleaginous diatoms, transcriptome analysis revealed unique expression patterns, such as concomitant upregulation of fatty acid/triacylglycerol biosynthesis and fatty acid degradation (β-oxidation) in concert with ATP production. This peculiar pattern of gene expression may account for the simultaneous growth and oil accumulation phenotype and may inspire novel biofuel production technology based on this oleaginous microalga.

Lipidomics and metabolomics communities comprise various informatics tools; however, software programs handling multimodal mass spectrometry (MS) data with structural annotations guided by the Lipidomics Standards Initiative are limited. Here, we provide MS-DIAL 5 for in-depth lipidome structural elucidation through electron-activated dissociation (EAD)-based tandem MS and determining their molecular localization through MS imaging (MSI) data using a species/tissue-specific lipidome database containing the predicted collision-cross section values. With the optimized EAD settings using 14 eV kinetic energy, the program correctly delineated lipid structures for 96.4% of authentic standards, among which 78.0% had the sn-, OH-, and/or C = C positions correctly assigned at concentrations exceeding 1 μM. We showcased our workflow by annotating the sn- and double-bond positions of eye-specific phosphatidylcholines containing very-long-chain polyunsaturated fatty acids (VLC-PUFAs), characterized as PC n-3-VLC-PUFA/FA. Using MSI data from the eye and n-3-VLC-PUFA-supplemented HeLa cells, we identified glycerol 3-phosphate acyltransferase as an enzyme candidate responsible for incorporating n-3 VLC-PUFAs into the sn1 position of phospholipids in mammalian cells, which was confirmed using EAD-MS/MS and recombinant proteins in a cell-free system. Therefore, the MS-DIAL 5 environment, combined with optimized MS data acquisition methods, facilitates a better understanding of lipid structures and their localization, offering insights into lipid biology.

Diatoms accumulate triacylglycerols in spherical organelles called oil bodies when exposed to nutrient deprivation conditions. Oil body biology in diatoms has attracted significant attention due to the complexity of the intracellular organelles and the unique combination of genes generated by the evolutionary history of secondary endosymbiosis. The demand for biofuel production has further increased the interest in and importance of a better understanding of oil body biology in diatoms, because it could provide targets for genetic engineering to further enhance their promising lipid accumulation. This review describes recent progress in studies of the structure and properties of diatom oil bodies. Firstly, the general features of diatom oil bodies are described, in particular, their number, size and morphology, as well as the quantity and quality of lipids they contain. Subsequently, the diatom oil body-associated proteins, which were recently discovered through oil body proteomics, are introduced. Then, the metabolic pathways responsible for the biogenesis and degradation of diatom oil bodies are summarized. During biogenesis and degradation, oil bodies interact with other organelles, including chloroplasts, the endoplasmic reticulum and mitochondria, suggesting their dynamic nature in response to environmental changes. Finally, the functions of oil bodies in diatoms are discussed. This article is part of the themed issue ‘The peculiar carbon metabolism in diatoms’.

Biofuel production using microalgae is believed to have the advantage of continuous year-round production over crop plants, which have strong seasonality. However, actual year-round production of microalgal lipids using outdoor mass cultivation has rarely been demonstrated. In our previous study, it was demonstrated that the oleaginous diatom, Fistulifera solaris, was culturable in outdoor bioreactors from spring to autumn, whereas biomass and lipid production in winter failed because F. solaris did not grow below 15 °C. Therefore, another candidate strain that is culturable in winter is required. In this study, a cold-tolerant diatom, Mayamaea sp. JPCC CTDA0820, was selected as a promising candidate for biofuel production in winter. Laboratory-scale characterization revealed that this diatom was culturable at temperatures as low as 10 °C. Subsequently, F. solaris (April–October) and Mayamaea sp. JPCC CTDA0820 (November–March) were cultured in outdoor open-pond bioreactors, wherein year-round production of diatom lipids was successfully demonstrated. The maximal values of areal productivities of biomass and lipids reached to 9.79 and 1.80 g/(m² day) for F. solaris, and 8.62 and 0.92 g/(m² day) for Mayamaea sp. JPCC CTDA0820, respectively. With the combined use of these two diatom species, stable year-round production of microalgal lipids became possible.

Microalgal oil is a promising alternative feedstock for biodiesel fuel (BDF). Mixotrophic cultivation with glycerol, the primary byproduct of BDF production, may be used to optimize BDF production. This strategy would reduce costs through glycerol recycling and improve lipid productivity and biomass productivity by overcoming the growth retardation caused by decreased light penetration in high-density culture. Overexpression of the endogenous glycerol kinase (GK) gene in an oleaginous marine diatom, Fistulifera solaris JPCC DA0580, accelerates glycerol metabolism and improves lipid and biomass productivities. Two candidates were selected from a collection of 90 G418-resistant clones, based on growth and confirmation of genome integration. GK gene expression was higher in the selected clones (GK1_7 and GK2_16) than in the wild-type culture. The GK2_16 clone achieved a 12% increase in lipid productivity. We have demonstrated the potential of metabolic engineering in oleaginous microalgae to improve lipid productivity. Metabolic engineering techniques can be used to optimize BDF production.