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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. [ABSTRACT FROM AUTHOR]

Oleaginous yeast Lipomyces starkeyi, a species in the Saccharomycetales order, has the capability to accumulate over 70% of its cell biomass as lipid under defined culture conditions. In this study, analysis of L. starkeyi AS 2.1560 proteome samples from different culture stages during a typical lipid production process was performed using an online multidimensional μRPLC/MS/MS method. Data searching against the proteome database of the yeast Saccharomyces cerevisiae led to the identification of 289 protein hits. Further comparative and semi-quantitative analysis under more stringent criteria revealed 81 proteins with significant expression-level changes. Among them, 52 proteins were upregulated and 29 proteins were downregulated. Gene ontology annotation indicated that global responses occurred when cells were exposed to the nitrogen deficiency environment for lipid production. Protein hits were annotated and largely concerned metabolic processes for alternative nitrogen sources usage or lipid accumulation. Many of the downregulated proteins were related to glycolysis, whereas the majority of the upregulated proteins were involved in proteolysis and peptidolysis, carbohydrate metabolism and lipid metabolism. Insights were provided in terms of cellular responses to nutrient availability as well as the basic biochemistry of lipid accumulation. This work presented potentially valuable information for understanding the biochemical events related to microbial oleaginity and rational engineering of oleaginous yeasts. [ABSTRACT FROM AUTHOR]