The basidiomycete yeast Rhodosporidium toruloides (also known as Rhodotorula toruloides) accumulates high concentrations of lipids and carotenoids from diverse carbon sources. It has great potential as a model for the cellular biology of lipid droplets and for sustainable chemical production. We developed a method for high-throughput genetics (RB-TDNAseq), using sequence-barcoded Agrobacterium tumefaciens T-DNA insertions. We identified 1,337 putative essential genes with low T-DNA insertion rates. We functionally profiled genes required for fatty acid catabolism and lipid accumulation, validating results with 35 targeted deletion strains. We identified a high-confidence set of 150 genes affecting lipid accumulation, including genes with predicted function in signaling cascades, gene expression, protein modification and vesicular trafficking, autophagy, amino acid synthesis and tRNA modification, and genes of unknown function. These results greatly advance our understanding of lipid metabolism in this oleaginous species and demonstrate a general approach for barcoded mutagenesis that should enable functional genomics in diverse fungi.

Glycosyltransferases are specific enzymes that catalyse the transfer of monosaccharide moieties to biological substrates, including proteins, lipids and carbohydrates. These enzymes are present from prokaryotes to humans, and their glycoconjugate products are often vital for survival of the organism. Many glycosyltransferases found in fungal pathogens such as Cryptococcus neoformans do not exist in mammalian systems, making them attractive potential targets for selectively toxic agents. In this article, we present the features of this diverse class of enzymes, and review the fungal glycosyltransferases that are involved in synthesis of the cell wall, the cryptococcal capsule, glycoproteins and glycolipids. We specifically focus on enzymes that have been identified or studied in C. neoformans, and we consider future directions for research on glycosyltransferases in the context of this opportunistic pathogen.

BackgroundThe filamentous fungus Neurospora crassa efficiently utilizes plant biomass and is a model organism for genetic, molecular and cellular biology studies. Here, a set of 567 single-gene deletion strains was assessed for cellulolytic activity as compared to the wild-type parental strain. Mutant strains included were those carrying a deletion in: (1) genes encoding proteins homologous to those implicated in the Saccharomyces cerevisiae secretion apparatus; (2) genes that are homologous to those known to differ between the Trichoderma reesei hyper-secreting strain RUT-C30 and its ancestral wild-type strain; (3) genes encoding proteins identified in the secretome of N. crassa when cultured on plant biomass and (4) genes encoding proteins predicted to traverse the secretory pathway.ResultsThe 567 single-gene deletion collection was cultured on crystalline cellulose and a comparison of levels of secreted protein and cellulase activity relative to the wild-type strain resulted in the identification of seven hyper-production and 18 hypo-production strains. Some of these deleted genes encoded proteins that are likely to act in transcription, protein synthesis and intracellular trafficking, but many encoded fungal-specific proteins of undetermined function. Characterization of several mutants peripherally linked to protein processing or secretion showed that the hyper- or hypo-production phenotypes were primarily a response to cellulose. The altered secretome of these strains was not limited to the production of cellulolytic enzymes, yet was part of the cellulosic response driven by the cellulase transcription factor CLR-2. Mutants implicated the loss of the SREBP pathway, which has been found to regulate ergosterol biosynthesis genes in response to hypoxic conditions, resulted in a hyper-production phenotype. Deletion of two SREBP pathway components in T. reesei also conferred a hyper-production phenotype under cellulolytic conditions.ConclusionsThese studies demonstrate the utility of screening the publicly available N. crassa single-gene deletion strain collection for a particular phenotype. Mutants in a predicted E3 ligase and its target SREBP transcription factor played an unanticipated role in protein production under cellulolytic conditions. Furthermore, phenotypes similar to those observed in N. crassa were seen following the targeted deletion of orthologous SREBP pathway loci in T. reesei, a fungal species commonly used in industrial enzyme production.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0297-9) contains supplementary material, which is available to authorized users.