Oleaginous yeasts are valuable systems for biosustainable production of hydrocarbon-based chemicals. Yarrowia lipolytica is one of the best characterized of these yeast with respect to genome annotation and flux analysis of metabolic processes. Nonetheless, progress is hampered by a dearth of genome-wide tools enabling functional genomics. In order to remedy this deficiency, we developed a library of Y. lipolytica insertion mutants via transposon mutagenesis. The Hermes DNA transposon was expressed to achieve saturation mutagenesis of the genome. Over 534,000 independent insertions were identified by next-generation sequencing. Poisson analysis of insertion density classified ~ 22% of genes as essential. As expected, most essential genes have homologs in Saccharomyces cerevisiae and Schizosaccharomyces pombe, and the majority of those are also essential. As an obligate aerobe, Y. lipolytica has significantly more respiration - related genes that are classified as essential than do S. cerevisiae and S. pombe. Contributions of non-essential genes to growth in glucose and glycerol carbon sources were assessed and used to evaluate two recent genome-scale models of Y. lipolytica metabolism. Fluorescence-activated cell sorting identified mutants in which lipid accumulation is increased. Our findings provide insights into biosynthetic pathways, compartmentalization of enzymes, and distinct functions of paralogs. This functional genomic analysis of the oleaginous yeast Y. lipolytica provides an important resource for modeling, bioengineering, and design of synthetic minimalized strains of respiratory yeasts.

Point mutations in Leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s disease (PD) and are implicated in a significant portion of apparently sporadic PD. Clinically, LRRK2-driven PD is indistinguishable from sporadic PD, making it an attractive genetic model for the much more common sporadic PD. In this review, we highlight recent advances in understanding LRRK2's subcellular functions using LRRK2-PD models, while also considering some of the limitations of these model systems. Recent developments of particular importance include new evidence of key LRRK2 functions in the endolysosomal system and LRRK2’s regulation of and by Rab GTPases. Additionally, LRRK2's interaction with the cytoskeleton allowed elucidation of the LRRK2 structure and appears relevant to LRRK2 protein degradation and LRRK2 kinase inhibitor therapies. We further discuss how LRRK2's interactions with other PD-driving genes, such as VPS35, GCase, and α-synuclein, may highlight cellular pathways more broadly disrupted in PD.

Yarrowia lipolytica is an oleaginous yeast that is recognized for its ability to accumulate high levels of lipids, which can serve as precursors to biobased fuels and chemicals. Polyketides, such as triacetic acid lactone (TAL), can also serve as a precursor for diverse commodity chemicals. This study used Y. lipolytica as a host organism for the production of TAL via expression of the 2-pyrone synthase gene from Gerbera hybrida. Induction of lipid biosynthesis by nitrogen-limited growth conditions increased TAL titers. We also manipulated basal levels of TAL production using a DNA cut-and-paste transposon to mobilize and integrate multiple copies of the 2-pyrone synthase gene. Strain modifications and batch fermentation in nitrogen-limited medium yielded TAL titers of 2.6 g/L. Furthermore, we show that minimal medium allows TAL to be readily concentrated at >94% purity and converted at 96% yield to pogostone, a valuable antibiotic. Modifications of this reaction scheme yielded diverse related compounds. Thus, oleaginous organisms have the potential to be flexible microbial biofactories capable of economical synthesis of platform chemicals and the generation of industrially relevant molecules.

Retroelement integration into host genomes affects chromosome structure and function. A goal of a considerable number of investigations is to elucidate features influencing insertion site selection. The Saccharomyces cerevisiae Ty3 retrotransposon inserts proximal to the transcription start sites (TSS) of genes transcribed by RNA polymerase III (RNAP3). In this study, differential patterns of insertion were profiled genome-wide using a random barcode-tagged Ty3. Saturation transposition showed that tRNA genes (tDNAs) are targeted at widely different frequencies even within isoacceptor families. Ectopic expression of Ty3 integrase (IN) showed that it localized to targets independent of other Ty3 proteins and cDNA. IN, RNAP3, and transcription factor Brf1 were enriched at tDNA targets with high frequencies of transposition. To examine potential effects of cis-acting DNA features on transposition, targeting was tested on high-copy plasmids with restricted amounts of 5′ flanking sequence plus tDNA. Relative activity of targets was reconstituted in these constructions. Weighting of genomic insertions according to frequency identified an A/T-rich sequence followed by C as the dominant site of strand transfer. This site lies immediately adjacent to the adenines previously implicated in the RNAP3 TSS motif (CAA). In silico DNA structural analysis upstream of this motif showed that targets with elevated DNA curvature coincide with reduced integration. We propose that integration mediated by the Ty3 intasome complex (IN and cDNA) is subject to inputs from a combination of host factor occupancy and insertion site architecture, and that this results in the wide range of Ty3 targeting frequencies.