Triacylglycerols are among the most attractive alternative raw materials for biofuel development. Current oil plant-based technologies are limited in terms of triacylglycerol production capacity and rate. These limitations may be circumvented by biotransformation of carbohydrates into lipids; however, our understanding of microbial oleaginicity remains limited. Here we present the results of a multi-omic analysis of Rhodosporidium toruloides, a robust triacylglycerol-producing fungus. The assembly of genome and transcriptome sequencing data reveals a genome of 20.2 Mb containing 8,171 protein-coding genes, the majority of which have multiple introns. Genes including a novel fatty acid synthase are predicted to participate in metabolic pathways absent in non-oleaginous yeasts. Transcriptomic and proteomic data suggest that lipid accumulation under nitrogen-limited conditions correlates with the induction of lipogenesis, nitrogenous compound recycling, macromolecule metabolism and autophagy. The multi-omic map of R. toruloides therefore provides a valuable resource for efforts to rationally engineer lipid-production pathways.

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The basidiomycetous yeast Rhodosporidium toruloides represents an excellent producer for microbial lipids and carotenoids. However, further rational engineering of this unconventional yeast remains challenging partially because of the absence of efficient and reliable transformation method. In this study, we developed an Agrobacterium-mediated transformation (ATMT) protocol for effective gene integration into the R. toruloides genome. Both haploid and diploid strains were successfully modified, and the integration was confirmed by colony PCR, Western blot analysis and genome walking. We further demonstrated that multiple genes could be integrated by consecutive ATMT, leading to engineered strains simultaneously resistant to multiple antibiotics. Our results provided a practical method for functional integration and expression of exogenous genes in R. toruloides, which should facilitate the development of genetic tools and the construction of superior strains to produce biofuel molecules and biochemicals.

BackgroundPlant volatiles play an important role in defending plants against insect attacks by attracting their natural enemies. For example, green leaf volatiles (GLVs) and terpenoids emitted from herbivore-damaged plants were found to be important in the host location of parasitic wasps. However, evidence of the functional roles and mechanisms of these semio-chemicals from a system of multiple plants in prey location by the parasitoid is limited. Little is known about the potential evolutionary trends between herbivore-induced host plant volatiles and the host location of their parasitoids.Methodology/Principal FindingsThe present study includes hierarchical cluster analyses of plant volatile profiles from seven families of host and non-host plants of pea leafminer, Liriomyza huidobrensis, and behavioral responses of a naive parasitic wasp, Opius dissitus, to some principal volatile compounds. Here we show that plants can effectively pull wasps, O. dissitus, towards them by releasing a universally induced compound, (Z)-3-hexenol, and potentially keep these plants safe from parasitic assaults by leafminer pests, L. huidobrensis. Specifically, we found that volatile profiles from healthy plants revealed a partly phylogenetic signal, while the inducible compounds of the infested-plants did not result from the fact that the induced plant volatiles dominate most of the volatile blends of the host and non-host plants of the leafminer pests. We further show that the parasitoids are capable of distinguishing the damaged host plant from the non-host plant of the leafminers.Conclusions/SignificanceOur results suggest that, as the most passive scenario of plant involvement, leafminers and mechanical damages evoke similar semio-chemicals. Using ubiquitous compounds, such as hexenol, for host location by general parasitoids could be an adaptation of the most conservative evolution of tritrophic interaction. Although for this, other compounds may be used to improve the precision of the host location by the parasitoids.

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NAD (NAD؉ ) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD ؉ level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in Escherichia coli cells that were genetically engineered to be NAD ؉ auxotrophic. First, we expressed the ntt4 gene encoding the NAD(H) transporter in the E. coli mutant YJE001, which had a deletion of the nadC gene responsible for NAD ؉ de novo biosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD ؉ . We then constructed the NAD ؉ -auxotrophic mutant YJE003 by disrupting the essential gene nadE, which is responsible for the last step of NAD ؉ biosynthesis in cells harboring the ntt4 gene. The minimal NAD ؉ level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD ؉ , while the maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD ؉ was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD ؉ concentration, until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044؋ and 9.6؋ those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed.

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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...