Rhodococcus opacus strain PD630 (R. opacus PD630), is an oleaginous bacterium, and also is one of few prokaryotic organisms that contain lipid droplets (LDs). LD is an important organelle for lipid storage but also intercellular communication regarding energy metabolism, and yet is a poorly understood cellular organelle. To understand the dynamics of LD using a simple model organism, we conducted a series of comprehensive omics studies of R. opacus PD630 including complete genome, transcriptome and proteome analysis. The genome of R. opacus PD630 encodes 8947 genes that are significantly enriched in the lipid transport, synthesis and metabolic, indicating a super ability of carbon source biosynthesis and catabolism. The comparative transcriptome analysis from three culture conditions revealed the landscape of gene-altered expressions responsible for lipid accumulation. The LD proteomes further identified the proteins that mediate lipid synthesis, storage and other biological functions. Integrating these three omics uncovered 177 proteins that may be involved in lipid metabolism and LD dynamics. A LD structure-like protein LPD06283 was further verified to affect the LD morphology. Our omics studies provide not only a first integrated omics study of prokaryotic LD organelle, but also a systematic platform for facilitating further prokaryotic LD research and biofuel development.

c Lipid droplets (LDs) are ubiquitous organelles that serve as a neutral lipid reservoir and a hub for lipid metabolism. Manipulating LD formation, evolution, and mobilization in oleaginous species may lead to the production of fatty acid-derived biofuels and chemicals. However, key factors regulating LD dynamics remain poorly characterized. Here we purified the LDs and identified LD-associated proteins from cells of the lipid-producing yeast Rhodosporidium toruloides cultured under nutrient-rich, nitrogen-limited, and phosphorus-limited conditions. The LD proteome consisted of 226 proteins, many of which are involved in lipid metabolism and LD formation and evolution. Further analysis of our previous comparative transcriptome and proteome data sets indicated that the transcription level of 85 genes and protein abundance of 77 proteins changed under nutrient-limited conditions. Such changes were highly relevant to lipid accumulation and partially confirmed by reverse transcription-quantitative PCR. We demonstrated that the major LD structure protein Ldp1 is an LD marker protein being upregulated in lipid-rich cells. When overexpressed in Saccharomyces cerevisiae, Ldp1 localized on the LD surface and facilitated giant LD formation, suggesting that Ldp1 plays an important role in controlling LD dynamics. Our results significantly advance the understanding of the molecular basis of lipid overproduction and storage in oleaginous yeasts and will be valuable for the development of superior lipid producers. Lipid droplets (LDs), intracellular organelles with deposits of neutral lipids and involved in many cellular activities, are widely present in both eukaryotic and prokaryotic cells (1-4). These organelles consist of a neutral lipid core surrounded by a phospholipid monolayer and associated proteins (3, 5). It has been known that LDs serve as the energy reservoir of cells, which may increase the adaptation by mobilization and degradation of lipids during nutrient deprivation, and also connect with other cellular processes, including lipid transport, membrane biogenesis, lipotoxicity relief, protein storage and degradation, pathogenicity, and autophagy (6-9). Because the biology of LDs is closely linked to some diseases, such as obesity, type 2 diabetes, and atherosclerosis, great progress has been made in elucidating the cellular trafficking, dynamics, and biogenesis of LDs in mammalian cells (2, 7, 10). However, there have been few studies on LDs in other species, especially naturally lipid-producing microorganisms (11-13). Analysis of these microorganisms is motivated by the fact that microbial lipid production holds a great promise to convert waste materials, including lignocellulosic biomass, into fatty acid-derived fuel molecules and chemicals in a scenario of biorefinery and sustainable development (14, 15).The major components of LDs are neutral lipids, including triacylglycerols (TAGs), sterol esters, and ether lipids (16). Neutral lipids constitute more than 90% of LDs by weight, but the ratio of TAGs to ste...