A molecular genetic toolbox for Yarrowia lipolytica
© The Author(s) 2017
Received: 24 June 2016
Accepted: 13 December 2016
Published: 3 January 2017
Yarrowia lipolytica is an ascomycete yeast used in biotechnological research for its abilities to secrete high concentrations of proteins and accumulate lipids. Genetic tools have been made in a variety of backgrounds with varying similarity to a comprehensively sequenced strain.
We have developed a set of genetic and molecular tools in order to expand capabilities of Y. lipolytica for both biological research and industrial bioengineering applications. In this work, we generated a set of isogenic auxotrophic strains with decreased non-homologous end joining for targeted DNA incorporation. Genome sequencing, assembly, and annotation of this genetic background uncovers previously unidentified genes in Y. lipolytica. To complement these strains, we constructed plasmids with Y. lipolytica-optimized superfolder GFP for targeted overexpression and fluorescent tagging. We used these tools to build the “Yarrowia lipolytica Cell Atlas,” a collection of strains with endogenous fluorescently tagged organelles in the same genetic background, in order to define organelle morphology in live cells.
These molecular and isogenetic tools are useful for live assessment of organelle-specific protein expression, and for localization of lipid biosynthetic enzymes or other proteins in Y. lipolytica. This work provides the Yarrowia community with tools for cell biology and metabolism research in Y. lipolytica for further development of biofuels and natural products.
KeywordsYarrowia lipolytica GFP localization Overexpression plasmid Genome sequence Tools Superfolder GFP Hygromycin B Protein tagging Organelle labeling Isogenic
Within a single fungal species, chromosome number and content can vary widely . Genomic instability has been documented in chromosomal changes in yeasts , induced in Candida albicans by centromere removal , and observed in the fragmentary gene order conservation of filamentous Ascomycetes . Yarrowia lipolytica also has evidence of inter-strain differences, demonstrated by CHEF gel . Studies in Y. lipolytica have primarily been done in three popular genetic backgrounds: W29 (Wild-type French strain ATCC20460™), H222 (wild-type German strain), and CBS6142-2 (the wild-type American strain) . The Po1 series, derived of a set of backcrosses between W29 and CBS6142-2 , have been used for a number of studies. CLIB122, or E150, the reference genome sequence, is derived of W29 in a cross with YB423-12, isolated from milled corn fiber tailings . Genome sequencing efforts have covered some original isolates and additional progeny from genetic studies, including strain W29 , and one of a backcrossed series, Po1f . Molecular genetic tools in Yarrowia include ablation of the ku70 ortholog, done in the citric acid producer H222, and in Po1d, which increases the rate of homologous recombination during transformation [11, 12]. Further genome sequencing is needed, as included for Po1g below, to clarify gene content and regulatory region differences between Y. lipolytica strains.
Yarrowia lipolytica has a precedence of organelle studies, particularly for peroxisome biogenesis and dynamics, including six stages of microbody development with differing size and contents . Study of catabolism in the peroxisome, and by lipases , modeling efforts [15, 16], and perturbation of both beta-oxidation and elements of the lipid biosynthetic pathways  have contributed to engineering desired products such as carotenoids , and omega-3 fatty acids . The availability of tools to identify organelle compartments would facilitate studies of this type.
A variety of stains are available for visualizing different intracellular compartments in yeast. FUN-1, Nile Red, MitoTracker, ER-Tracker, and DAPI among others can be used to visualize the vacuole, lipid droplet, mitochondria, endoplasmic reticulum, and nucleus, respectively. Compendia of cell staining techniques are available for specific organisms  or specific organelles [21, 22]. However, in Y. lipolytica, we observed poor penetration, toxicity, the need for fixation, and resistance to stain uptake, consistent with many fungi [23–26]. Fixation frequently alters intracellular morphology and staining effectiveness when using these stains. Similarly, changes in growth conditions, or incubation buffer may perturb stain application, as stain efflux differences can be pH dependent . The cell wall changes thickness in an age- and nutrient-dependent manner , and may provide an additional barrier to permeability. Probe or stain molecule size requiring a chemical vehicle which increases permeability of cell membranes (e.g., DMSO) may also affect cell health and cogency of data, just as complications of dye delivery under nutrient specific conditions and removal of excess dye may perturb cell integrity. Organelle-specific proteins tagged with green fluorescent protein (GFP) overcome many of these limitations, particularly for life cell imaging.
The technique of using GFP labeled proteins for localization and expression is more than two decades old . The Yarrowia community has developed plasmids for over-expression, or carbon-induced expression using a non-integrating cassette for secretion or exogenous protein expression [7, 30, 31]. Their use is dependent upon having the correct auxotrophs and efficient transformation methods [17, 32]. Fluorescent protein studies in Y. lipolytica have encompassed tagging for localization of Fat1p, Fat4p, and Faa1p using plasmids [33, 34]. Similarly, hybrid promoter studies have utilized fluorescent proteins , and transcription factors have been localized using a GFP-fusion expressed from a plasmid . However, sets of Y. lipolytica strains with GFP tagged organelles are not available.
Tools presented here will allow definition of pathways, localization of biosynthetic enzymes, and organelle dynamics in living cells. We developed an isogenic ku70::hph strain set for improved homologous recombination efficiency when transforming PCR products, and assessing localization of proteins within a cell by fluorescent tagging under a high expression promoter using auxotrophic selection of transformants or integrants. This genetic background was sequenced and annotated to facilitate genetic studies. A superfolder GFP gene which shows bright fluorescence , was codon optimized for multi-modal use in Y. lipolytica, demonstrated by an enzyme over-expression collection using lipid biosynthesis proteins. To query organelle dynamics in response to changing environmental conditions and build a tool set for co-localization studies, we built the Y. lipolytica Cell Atlas composed of seven strains with different cell compartment labels in both auxotrophic and prototrophic backgrounds. This work provides a consistent set of strains and tools for genetics and cell biology in Y. lipolytica and demonstrates the dynamic nature of organelles important for energy metabolism under conditions relevant to industrial biofuel production.
Results and discussion
Construction of isogenic NHEJ-deficient auxotrophic strains
Strains used in this study
Gaillardin et al. 
matA, xpr2-332, axp-2, leu2-270
Madzak et al. 
matA, xpr2-332, axp-2, ku70::hph+, leu2-270
matA, xpr2-332, axp-2, ku70::hph+, leu2-270::leu2+
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, ura3
matA, xpr2-332, axp-2, ku70::hph+, leu2-270::leu2+, ura3
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, erg6-sfGFP:leu2+
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, pex13-sfGFP:leu2+
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, hH1-sfGFP:leu2+
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, rdl2-sfGFP:leu2+
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, aim17-sfGFP:leu2+
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, emc2-sfGFP:leu2+
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, cpy1-sfGFP:leu2+
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, arx1-sfGFP:leu2+
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, vrg4-sfGFP:leu2+
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, erg6-sfGFP:leu2+, ura3
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, pex13-sfGFP:leu2+, ura3
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, hH1-sfGFP:leu2+, ura3
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, rdl2-sfGFP:leu2+, ura3
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, aim17-sfGFP:leu2+, ura3
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, emc2-sfGFP:leu2+, ura3
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, cpy1-sfGFP:leu2+, ura3
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, arx1-sfGFP:leu2+, ura3
matA, xpr2-332, axp-2, ku70::hph+, leu2-270, vrg4-sfGFP:leu2+, ura3
Genome sequencing, assembly, and annotation of NHEJ-deficient base strain
New transcripts by RNA-seq in FKP355 compared to CLIB122
Transcript ID to CLIB122 (%)
Metal dependant phosphohydrolase
Ribosomal protein L15
Exonuclease domain containing protein
Retrotransposon ty3-gypsy subclass
Orotidine 5-phosphate decarboxylase (ura3)
Kelch repeat protein
Clathrin coat assembly protein
Conserved hypothetical protein
Conserved hypothetical protein
Serine threonine protein phosphatase
Protein with serine active lipase domain
Mfs general substrate transporter
Putative cation transporter
Cytochrome p450 alkane
Construction of a multipurpose vector for rapid expression of fluorescently tagged proteins in Yarrowia lipolytica
Expression of sfGFP-tagged lipid biosynthetic enzymes reveals their localization
Localization of Yarrowia and Saccharomyces lipid biosynthetic enzymes
Y. lipolytica gene
S. cerevisiae homolog
Construction of an atlas of endogenous sfGFP-tagged organelles
Yarrowia identity and coverage compared to S288C proteins
Mitochondrial response to reactive oxygen species is growth phase dependent
We wanted to assess the response of Rdl2p to hydrogen peroxide stress. It was previously observed that S. cerevisiae sensitivity to hydrogen peroxide is enhanced in minimal medium compared with the presence of amino acids and nucleobases , a susceptibility also confirmed in Y. lipolytica . Further, it has been noted that prior exposure of yeast to an oxidative stress increases tolerance to higher concentrations of ROS (reactive oxygen species)  suggesting a physiological response which may be based in protein expression. ROS tolerance is also increased in stationary vs. exponential phase cells . Susceptibility to and tolerance of oxidative stress is relevant to lipid biogenesis in several respects: lipids are a target for peroxidation by ROS, and the oxidative state of the cell may change the level of available NADPH, which is used directly in lipid biogenesis.
Pex13-sfGFPp signal is altered by carbon source and nitrogen limitation
The dynamics of Y. lipolytica peroxisomes have been followed by immunostaining [101, 102], by biochemical sedimentation gradients [13, 103], and mass spectrometry . Expression of a limited number of peroxisomal protein components have been developed in other backgrounds [105, 106]. However, previously studied proteins are involved in beta-oxidation (Mfe1p), or are not a constant peroxisome resident (Pex3p) . Malate synthase has been studied for alternative splicing, with one form localized by an N-terminal GFP to the peroxisome matrix by plasmid-based expression . Peroxisome maturation has been studied in detail in Y. lipolytica, following a series of microbody structures which contain peroxisome associated proteins (Pex6p and Pex2p) and enzyme functions such as beta-oxidation and hydrogen peroxide detoxification . Loss of peroxisome-localized beta-oxidation enzyme function resulting in increased lipid accumulation shows the importance of this organelle to biofuel development .
The cell biology of lipid accumulation is of industrial interest. Here, we have developed a suite of isogenic strains and codon optimized plasmids to enable cell biology studies in Y. lipolytica. We have sequenced, assembled, and annotated the initial non-homologous end-joining mutant strain used for construction of all the strains described herein allowing for accurate design of genetic constructs. Although some of the resources we have developed have been previously described in other genetic backgrounds in Y. lipolytica , including auxotrophs , utilization of hph  and gfp , and replicating and integrating plasmids, they have guided the design of these strains and plasmids. Having all these tools, along with the fluorescent organelle atlas in an isogenic non-homologous end-joining mutant will remove data differences caused by genetic background, and expedite functional genetics in Y. lipolytica. Organelle-specific GFP tagging can identify specific membrane structures under conditions relevant to industrial use, and parallel analysis in an isogenic background with live GFP-labeled proteins expressed from endogenous loci to monitor the size, shape, and quantity of a particular organelle. The atlas strains presented allow rapid and efficient targeted disruption, tagging, overexpression, multi-protein labeling with additional fluorescent tags, and an indicator of cellular stress as suggested by Rdl2-sfGFPp expression in response to reactive oxygen species (Fig. 10). Biofuel organism engineering efforts require consideration not only of total lipid accumulation, but also of cell health and growth rate. Continual drain on lipid resources may make senescence an issue, as the balance of triglyceride synthesis has been shown to confer an energy dependence increase in longevity to S. cerevisiae .
We present these tools as a compliment to the holistic approach on the global level of –omic analyses. The rapid increase in –omic studies in Y. lipolytica, which inquire multiple levels of regulation, has generated many hypotheses [15, 28, 117–119] that require more efficient tools for functional genomics. For example, during lipid accumulation experiments, membrane bound cellular substructures can be distinguished by confocal microscopy. While lipid droplets are frequently notable due to a difference in bright field diffraction, quantifying and evaluating their growth in live cells are a GFP functionality of interest for exploring lipid and metabolic phenotypes. We found previously that protein degradation, peroxisome activity, and alcohol sugar secretion were characteristics of Y. lipolytica during nitrogen starvation . In order to study compartmentalized activities, we have included atlas strains demarking membrane-bound compartments relevant to these processes, including the vacuole (nitrogen storage and material degradation), mitochondria (producer of ATP and NADH and NADPH for lipid biosynthesis), golgi (protein modification, sorting, and secretion), peroxisome (oxidative detoxification, beta-oxidation), nucleus (transcriptional activity), and the endoplasmic reticulum (translation and lipid droplet biogenesis) which will enable new routes for the investigation of energy relevant organelles by the Yarrowia research community.
All chemicals and reagents were purchased from Thermo Fisher Scientific (Waltham, MA) unless otherwise noted. The water used was of Milli-Q grade purified by a Millipore (Bedford, MA) Milli-Q UV Purification System. PCR amplification was performed using Q5 high fidelity DNA polymerase (New England Biolabs; Ipswich, MA). Digestions used FastDigest enzymes (Thermo Fisher Scientific; Waltham, MA) and ligations used T4 DNA ligase (Life Tech.; Carlsbad, CA).
Yeast strains and cultivation
Strains were maintained on YPD medium (1% yeast extract, 1% peptone, 2% glucose) or YNB medium (1.71 g/L Yeast Nitrogen base lacking amino acids, 5 g/L ammonium sulfate, 5 g/L glucose) at 28 °C unless otherwise noted. The widely used Y. lipolytica strain Po1g  is from Yeastern Biotech (Taipei, Taiwan). Wild-type matA (W29; ATCC20460™) and matB (CBS6124-2; ATCC18944™) Y. lipolytica strains were from American Type Tissue Culture (Manassas, VA).
Generation of ku70::hph construct
To create the ku70 gene deletion construct, 1.9 kb upstream and downstream of the ku70 gene and 0.55 kb of the Y. lipolytica tef1 promoter and the bacterial hygromycin phosphotransferase gene (hph) were amplified by PCR with oligo pairs (Additional file 3, Oligos) 1313/1314 (ku70 upstream), 1315/1321 (hph), 1322/1318 (tef1), and 1319/1320 (ku70 downstream), respectively. During PCR amplification, restriction sites for the endonucleases XhoI and HindIII were introduced at the 5′-end of ku70 upstream and 3′-end of ku70 downstream for further cloning. All four fragments were fused together by yeast gap repair with pRS426 vector as previously described  and then cloned into the T-DNA binary vector pZD663 (see Additional file 1: Figure S2). The fragment was confirmed by DNA sequencing. The pZD663 binary vector, a derivative of the pBI121 binary Ti plasmid , was constructed by replacing the whole DNA fragment between left and right borders of the pBI121 T-DNA region (GenBank: AF485783.1) with a synthetic DNA fragment containing ten unique multiple cloning sites.
The transgene expression T-DNA binary vector pZD663-ku70 downstream-tef1-hph-ku70 upstream was mobilized into the A. tumefaciens EHA105 strain by a freeze–thaw technique . A. tumefaciens was grown in YEP (10 g/L yeast extract, 10 g/L peptone and 5 g/L NaCl) overnight. Agrobacterium cells were aliquoted into 5-mL induction medium  with and without 0.2 mM AS (acetosyringone) to a final density of 0.2 at A600 for an additional 5–6 h growth to final OD600 of 0.4–0.5 (~2 × 109 cells/mL determined by plate growth count). Three different amounts (5 × 106, 1 × 107, and 5 × 107 cells) of overnight Y. lipolytica cells grown in YPD medium were aliquoted into microcentrifuge tubes and washed twice with IM buffer. Five different ratios of Y. lipolytica and Agrobacterium cells (i.e., 5 × 106, 1 × 107, and 5 × 107 Y. lipolytica:100 µL (~2 × 108) Agrobacterium cells with 0.2 mM AS; 1 × 107 Y. lipolytica:300 µL (~6 × 108) of Agrobacterium cells with 0.2 mM AS; and 1 × 107 Y. lipolytica:100 µL (~2 × 108) of Agrobacterium cells without AS) were mixed well in a final volume of 200 µL and spread onto the 25 × 30 mm sterile 0.45 µm Hybond-N + nylon membrane (GE Healthcare Bio-Sciences, Pittsburgh, Pennsylvania) laid on the IM agar plate with or without AS. After 2 days of incubation at room temperature (~23 °C), the transformed Y. lipolytica cells were washed from the nylon membrane with 5 mL sterile distilled water and 1/10 of the volume was spread onto the YPD agar plate with the 300 mg/L hygromycin B and 250 mg/L cefotaxime. The transformed Y. lipolytica cells were visible on the plate after incubation at 28 °C for 2 days. Individual colonies were picked and streaked onto a new YPD agar plate containing 300 mg/L hygromycin B and 250 mg/L cefotaxime (see Additional file 1: Figure S1). Single colonies were grown in 2 mL of YPD liquid medium containing the same set of antibiotics at 28 °C and 200 rpm for 18–24 h, which was used for strain storage and genomic DNA isolation.
Identification of Y. lipolytica ku70::hph clones
The genomic DNA of selected Y. lipolytica transgenic clones was isolated by CTAB method  with modifications. Overnight cultures were transferred into a 2-mL screw-cap micro-tubes (Mikro-Schraubrohre) and centrifuged for 2 min at 17,000g and 20 °C. The supernatant was removed and 250 μL of CTAB and 250 µL of 0.5 mm zirconia/silica beads were added to each tube. The cells were homogenized in a mini-beadbeater (Biospec) for 2 min. The homogenized cell mixtures were incubated at 58 °C for 1 h. Genomic DNA was extracted with 170 µL of phenol:chloroform:isoamyl alcohol (25:24:1, pH 8.0) once and centrifuged for 8 min at 17,000g. After transfer, 500 μL of 95% ethanol was mixed with the supernatants, and incubated at room temperature for 10 min. The DNA was pelleted at 17,000g and 15 °C for 10 min, then re-suspended in 200 µL of 50× TE (50 mM TrisHCl/10 mM EDTA, pH8.0) containing 20 µg of RNase A per sample and incubated at 55 °C for 30~60 min. The DNA mixture was extracted twice with 200 µL of phenol:chloroform:isoamyl alcohol and centrifuged at 17,000g for 10 min. For precipitation, 20 µL of 3 M sodium acetate (pH 5.2) and 500 μL of 95% ethanol were added to each DNA sample, mixed gently and kept at room temperature for 10~30 min. The genomic DNA was pelleted by centrifugation at 10,000g and 15 °C for 10 min, then washed once with 1 mL of 70% ethanol. The dried DNA pellets were re-suspended with 50 µL of 10 mM Tris–HCl (pH 8.0). The genomic DNA was used for PCR screening with oligo pairs located outside the transgene deletion fragment of ku70 (see Additional file 1: Figure S2B). The PCR product was digested with PvuII and separated by agarose gel electrophoresis to test for ku70 gene replacement by hph. After screening about 120 individual transformants, we identified a clone in which ku70 was replaced by hph (see Additional file 1: Figure S2C).
Southern blotting analysis
For Southern blotting, one microgram of total genomic DNA from Po1g or ku70::hph strains was digested with the restriction endonuclease BglII, EcoRV, and PvuII, respectively. The genomic DNA fragments were separated by electrophoresis in a 1% agarose gel, then transferred onto the Amersham Hybond-N+ membrane (GE Healthcare Life Sci, Pittsburgh, PA) by alkaline capillary transfer. The 2.0 kb genomic DNA fragment of ku70 gene upstream region used for the ku70 deletion construct was used to prepare a biotin-labeled probe (see Additional file 1: Figure S2D). The genomic DNA on the Hybond-N+ membrane was hybridized with the biotin-labeled probe overnight at 60 °C in a Problot Hybridization Oven (Labnet International, Edison, NJ, USA) and visualized with a North2South chemiluminescent detection kit (Pierce Protein Research Products, Rockford, IL) in a Koda Imaging Station 2000R (Eastman Kodak Company, Rochester, NY, USA).
Generation of isogenetic ku70::hph auxotrophs
Ura3 mutants derived from FKP355 were selected on YNB with 5 g/L uracil and 5 g/L 5-FOA, and supplemented with 0.1 g/L leucine to make FKP393. Leu2 was complemented in FKP355 resulting in FKP391 and also in FKP393, yielding FEB130 by transformation with a leu2 PCR product amplified with primers OKP443 and OKP444.
Genome sequencing, assembly, and annotation
Genomic DNA was isolated from FKP355 using the yeast genomic DNA purification kit (AMRESCO, Solon, OH) followed by 150 bp paired-end sequencing on an Illumina MiSeq instrument. The reads were assembled using Velvet v1.2.10  with a k-mer length of 81 chosen to optimize for the highest N 50. Raw RNA-seq data from previously described strains of similar genetic background [15, 51] were mapped to the assembled genome using TopHat v2.0.13  to predict transcripts from which coding regions were predicted. Predicted proteins were functionally annotated using Blast2GO . Full length transcripts were aligned to contigs from strain CLIB122  using Blat v32×1  to determine their position and percent identity. Reads were aligned to the reference genome using Bowtie v0.12.9  and SNPs identified with custom Perl scripts.
A codon optimized superfolder GFP (sfGFP) gene  followed by a codon optimized hph gene from Escherichia coli  driven by the high expression Yarrowia lipolytica tef promoter along with the centromere and origin of replication sequences from ARS68  were synthesized and cloned into pMK-RQ to make pYL1. The high expression exp1 promoter was amplified from Y. lipolytica strain Po1g with primer pairs OKP189/190 and ligated into pYL1 following digestion with KpnI and EcoRI to make pYL2 (Genbank: KU378203). The Y. lipolytica leu2 gene was amplified with primers OKP193/194 and ligated into pYL2 following digestion with BamHI to make plasmid pYL4. Additional leu2 5′ flanking sequence was amplified with primer pair OKP451/452 and ligated into pYL4 following digestion with HindIII to make pYL15 (Genbank: KU378202). Homologs of enzymes predicted to be directly involved in the biosynthesis of triglycerides from glucose were predicted using BlastP. The coding region for each enzyme was PCR amplified from Y. lipolytica strain FKP355 genomic DNA using Q5 DNA polymerase (New England Biolabs, Ipswich, MA) and oligos (Life Technologies, Carlsbad, CA) designed with 5′ (5′-ATATCTACAGCGGTACCCCC-3′) and 3′ (5′-CCGCCTCCGCCGATATCCCCC-3′) overhangs homologous to plasmid pYL15 (listed in Additional file 3). Plasmid pYL15 digested with SmaI (Fermentas, Waltham, MA) and the PCR products were purified using a GeneJET purification kit (Thermo Fisher Scientific, Waltham, MA) and assembled using the NEBuilder HiFi assembly kit (New England Biolabs, Ipswich, MA) to produce the replicating plasmids listed in Additional file 4. FKP355 was transformed with pYL15 derived plasmids using the lithium acetate method  followed by selection on YNB agar. Transformants were verified by PCR and microscopy and maintained at −80 °C in 15% glycerol.
Generation of organelle GFP library
Selection of candidate organelle-targeted proteins was done using quantitative and image data in S. cerevisiae GFP libraries [77, 78, 132]. For transformation, we utilized a PEG-lithium acetate buffer method developed for S. cerevisiae with modifications . Y. lipolytica cells were grown for 1-3 days before transformation. DNA fragments of approximately 1 kb upstream (5′ flank) and downstream (3′ flank) of the stop codon of the gene for C-terminal GFP tagging were amplified (oligos used in Additional file 3). The leu2 gene was amplified from pYL4 using OEB 170/171, and the codon optimized sfGFP from pYL2 using OKP31/OEB169 which append a synthetic linker for attachment to the selective gene. Targeted proteins selected by orthologous Blast (Table 4). Overlap PCRs were used to assemble the 5′ flank and sfGFP plus a partial region in the selection gene, and separately, another portion of the selection gene and the 3′ flank (OEB3/4 or OKP 211/212). These extension PCRs were run using a 15-cycle program to allow the fragments to prime against one another (98 for 2:00, with cycles of 98 for 0:30, 60 for 0:20, 72 for 2:00, followed by 72 for 5:00). Primers were added (5′ flank forward or 3′ flank reverse primer with the appropriate split marker primer), and the PCRs continued for 30 cycles. Gel purified fragments were extracted using a GeneJET Micro kit (K0832). Agar plates containing YNB with appropriate Leucine or Uracil supplementation (5 g/L) were used for selection [131, 133]. For organelle response to nitrogen limitation, strains were grown to turbidity shaking in YPD for 16 h (t0) followed by washing in YNB salts, and transfer to Y-D media lacking a nitrogen source for 20 h (t1).
For microscopy, cells were visualized using a Zeiss LSM710 confocal laser-scanning microscope (Carl Zeiss MicroImaging GmbH, Munchen, Germany) with a Plan-Apochromate 100×/1.4 Oil objective. For co-localization studies, the cell cultures were stained with MitoTracker deep red (Molecular Probes-Thermo-Fisher, M22426, Eugene, OR) or lipidTOX red (Molecular Probes) according to the manufacturer’s instructions. Cells were grown overnight in 4 mL of YPD in a shake tube, then 700 μL was pelleted gently and transferred to 2 mL fresh YPD for 1 h containing MitoTracker Deep Red added to a 1:5000 dilution. Calcofluor white was added to the cells at the time of imaging with the addition of 1 μL of 1:150 dilution of a 1 mg/mL stock solution to every 4 μL of cells. Images were processed using imageJ  and CellProfiler . Images were normalized within the tagged strain relative to wild-type or within time point as appropriate.
Hydrogen peroxide stress
Atlas strains FEB64, FEB92, FEB93 were grown 16 h in YPD with shaking. For exponential phase cells, 1 mL of culture was transferred to fresh media, while the remaining stationary cells were used in parallel at the 24 h mark for stress treatment. Briefly, cells were collected by centrifugation at 200 rpm for 1 min. Reserving some of the initial culture for imaging (t0), strains were transferred to YPD containing 40 mM hydrogen peroxide, then imaged at 1–2 h (t1) and 20 h (t2).
Peroxisome media assay
Strain FEB64 was grown overnight, collected by centrifugation, washed 1X in PBS, and then equally inoculated into 10 mL of rich (YPD), minimal (YNB; 1.7 g/L yeast nitrogen base without amino acids and ammonium sulfate, 20 g/L glucose, 5 g/L ammonium sulfate), nitrogen-deficient rich (Y-D; 10 g/L yeast extract, 20 g/L glucose) and nitrogen-deficient minimal medium (YNBnoN; 1.7 g/L yeast nitrogen base without amino acids and ammonium sulfate, 20 g/L glucose). Cultures were grown shaking at 200 rpm for 16 h, and then imaged. Calcofluor staining was used for definition of cellular space in Cell Profiler.
green fluorescent protein
superfolder green fluorescent protein
FEB-fungal strain numbers by author initials
hygromycin phosphotrasferase, gene confers resistance to hygromycin B
peroxisome targeting signal
reactive oxygen species
single nucleotide polymorphism
yeast extract, peptone, dextrose media, see “Methods” section
yeast nitrogen base media, see “Methods” section
ELB, KRP, ZD, EJK, JN, and SEB conceived and designed the experiments, and wrote the paper. ELB, SEB, KRP, ZD performed the experiments and analyzed the data. All authors read and approved the final manuscript.
Part of this research was performed at the Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility sponsored by the U.S. DOE OBER, and located at PNNL. PNNL is a multiprogram national laboratory operated by Battelle for the DOE under Contract DE-AC05-76RLO 1830.
The authors declare that they have no competing interests.
Availability of data and materials
Yarrowia lipolytica FKP355, FEB130, FKP391, FKP393, and atlas strains are available at the Fungal Genetics Stock Center (Manhattan, KS, http://www.fgsc.net).
Plasmid sequences are available for pYL2 (GenBank Accession: KU378203), and pYL15 (GenBank Accession: KU378202).
This material is based upon work supported by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research (OBER), Genomic Science program, under Award Number DE-SC0008744. Support was also provided by a William Wiley postdoctoral fellowship.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Vlaardingerbroek I, Beerens B, Rose L, Fokkens L, Cornelissen BJC, Rep M. Exchange of core chromosomes and horizontal transfer of lineage-specific chromosomes in Fusarium oxysporum: chromosome transfer and exchange in F. oxysporum. Environ Microbiol. 2016. doi:10.1111/1462-2920.13281.Google Scholar
- Gordon JL, Byrne KP, Wolfe KH. Mechanisms of chromosome number evolution in yeast. PLoS Genet. 2011;7:e1002190 (Fay JC, editor).View ArticleGoogle Scholar
- Ketel C, Wang HSW, McClellan M, Bouchonville K, Selmecki A, Lahav T, et al. Neocentromeres form efficiently at multiple possible loci in Candida albicans. PLoS Genet. 2009;5:e1000400 (Copenhaver GP, editor).View ArticleGoogle Scholar
- Hane JK, Rouxel T, Howlett BJ, Kema GH, Goodwin SB, Oliver RP. A novel mode of chromosomal evolution peculiar to filamentous Ascomycete fungi. Genome Biol. 2011;12:R45.View ArticleGoogle Scholar
- Naumova E, Naumov G, Fournier P, Nguyen HV, Gaillardin C. Chromosomal polymorphism of the yeast Yarrowia lipolytica and related species: electrophoretic karyotyping and hybridization with cloned genes. Curr Genet. 1993;23:450–4.View ArticleGoogle Scholar
- Beopoulos A, Desfougeres T, Sabirova J, Zinjarde S, Neuvéglise C, Nicaud J-M. The hydrocarbon-degrading oleaginous yeast Yarrowia lipolytica. In: Timmis KN, editor. Handb. Hydrocarb. Lipid Microbiol. [Internet]. Berlin, Heidelberg: Springer Berlin Heidelberg; 2010. p. 2111–2121. http://link.springer.com/10.1007/978-3-540-77587-4_152. Accessed 10 Jun 2016.
- Madzak C, Tréton B, Blanchin-Roland S. Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica. J Mol Microbiol Biotechnol. 2000;2:207–16.Google Scholar
- Wickerham LJ, Kurtzman CP, Herman AI. Sexual reproduction in Candida lipolytica. Science. 1970;167:1141.View ArticleGoogle Scholar
- Pomraning KR, Baker SE. Draft genome sequence of the dimorphic yeast Yarrowia lipolytica strain W29. Genome Announc. 2015;3:e01211–5.View ArticleGoogle Scholar
- Liu L, Alper HS. Draft genome sequence of the oleaginous yeast Yarrowia lipolytica PO1f, a commonly used metabolic engineering host. Genome Announc. 2014;2:e00652-14.View ArticleGoogle Scholar
- Kretzschmar A, Otto C, Holz M, Werner S, Hübner L, Barth G. Increased homologous integration frequency in Yarrowia lipolytica strains defective in non-homologous end-joining. Curr Genet. 2013;59:63–72.View ArticleGoogle Scholar
- Verbeke J, Beopoulos A, Nicaud J-M. Efficient homologous recombination with short length flanking fragments in Ku70 deficient Yarrowia lipolytica strains. Biotechnol Lett. 2013;35:571–6.View ArticleGoogle Scholar
- Titorenko VI, Chan H, Rachubinski RA. Fusion of small peroxisomal vesicles in vitro reconstructs an early step in the in vivo multistep peroxisome assembly pathway of Yarrowia lipolytica. J Cell Biol. 2000;148:29–44.View ArticleGoogle Scholar
- Fickers P, Marty A, Nicaud JM. The lipases from Yarrowia lipolytica: genetics, production, regulation, biochemical characterization and biotechnological applications. Biotechnol Adv. 2011;29:632–44.View ArticleGoogle Scholar
- Kerkhoven EJ, Pomraning KR, Baker SE, Nielsen J. Regulation of amino-acid metabolism controls flux to lipid accumulation in Yarrowia lipolytica. Npj Syst Biol Appl. 2016;2:16005.View ArticleGoogle Scholar
- Kavšček M, Bhutada G, Madl T, Natter K. Optimization of lipid production with a genome-scale model of Yarrowia lipolytica. BMC Syst Biol [Internet]. 2015; 9. http://www.biomedcentral.com/1752-0509/9/72. Accessed 15 Jan 2016.
- Blazeck J, Hill A, Liu L, Knight R, Miller J, Pan A, et al. Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production. Nat Commun [Internet]. 2014; 5. http://www.nature.com/doifinder/10.1038/ncomms4131. Accessed 15 Jan 2016.
- Matthaus F, Ketelhot M, Gatter M, Barth G. Production of lycopene in the non-carotenoid-producing yeast Yarrowia lipolytica. Appl Environ Microbiol. 2014;80:1660–9.View ArticleGoogle Scholar
- Xue Z, Sharpe PL, Hong S-P, Yadav NS, Xie D, Short DR, et al. Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica. Nat Biotechnol. 2013;31:734–40.View ArticleGoogle Scholar
- Wolinski H, Kohlwein SD. Single yeast cell imaging. Methods Mol Biol Clifton NJ. 2014;1205:91–109.View ArticleGoogle Scholar
- Dimmer KS. Fluorescence Staining of Mitochondria for morphology analysis in Saccharomyces cerevisiae. In: Xiao W, editor. Yeast Protoc [Internet]. New York: Springer New York; 2014. p. 131–52. http://link.springer.com/10.1007/978-1-4939-0799-1_9. Accessed 28 Mar 2016.
- Wolinski H, Kohlwein SD. Microscopic and spectroscopic techniques to investigate lipid droplet formation and turnover in yeast. Methods Mol Biol Clifton NJ. 2015;1270:289–305.View ArticleGoogle Scholar
- Chaffin WL, López-Ribot JL, Casanova M, Gozalbo D, Martínez JP. Cell wall and secreted proteins of Candida albicans: identification, function, and expression. Microbiol Mol Biol Rev. 1998;62:130–80.Google Scholar
- Ram AFJ, Klis FM. Identification of fungal cell wall mutants using susceptibility assays based on Calcofluor white and Congo red. Nat Protoc. 2006;1:2253–6.View ArticleGoogle Scholar
- Gulshan K, Moye-Rowley WS. Multidrug resistance in fungi. Eukaryot Cell. 2007;6:1933–42.View ArticleGoogle Scholar
- Fotopoulos V. Never say dye: new roles for an old fluorochrome. Plant Signal Behav. 2012;7:342–4.View ArticleGoogle Scholar
- Aguedo M, Waché Y, Belin J-M. Intracellular pH-dependent efflux of the fluorescent probe pyranine in the yeast Yarrowia lipolytica. FEMS Microbiol Lett. 2001;200:185–9.View ArticleGoogle Scholar
- Pomraning KR, Wei S, Karagiosis SA, Kim Y-M, Dohnalkova AC, Arey BW, et al. Comprehensive metabolomic, lipidomic and microscopic profiling of Yarrowia lipolytica during lipid accumulation identifies targets for increased lipogenesis. PLOS ONE. 2015;10:e0123188 (Nowrousian M, editor).View ArticleGoogle Scholar
- Chalfie M, Tu Y, Euskirchen G, Ward W, Prasher D. Green fluorescent protein as a marker for gene expression. Science. 1994;263:802–5.View ArticleGoogle Scholar
- Nicaud J-M, Madzak C, van den Broek P, Gysler C, Duboc P, Niederberger P, et al. Protein expression and secretion in the yeast Yarrowia lipolytica. FEMS Yeast Res. 2002;2:371–9.Google Scholar
- Barth G, Gaillardin C. Yarrowia lipolytica. Nonconv Yeasts Biotechnol [Internet]. Berlin, Heidelberg: Springer Berlin Heidelberg; 1996. p. 313–88. http://www.springerlink.com/index/10.1007/978-3-642-79856-6_10. Accessed 4 May 2016.
- Wang Z-P, Xu H-M, Wang G-Y, Chi Z, Chi Z-M. Disruption of the MIG1 gene enhances lipid biosynthesis in the oleaginous yeast Yarrowia lipolytica ACA-DC 50109. Biochim Biophys Acta BBA-Mol Cell Biol Lipids. 2013;1831:675–82.View ArticleGoogle Scholar
- Dulermo R, Gamboa-Meléndez H, Dulermo T, Thevenieau F, Nicaud J-M. The fatty acid transport protein Fat1p is involved in the export of fatty acids from lipid bodies in Yarrowia lipolytica. FEMS Yeast Res. 2014;14:883–96.View ArticleGoogle Scholar
- Tenagy, Park JS, Iwama R, Kobayashi S, Ohta A, Horiuchi H, et al. Involvement of acyl-CoA synthetase genes in n-alkane assimilation and fatty acid utilization in yeast Yarrowia lipolytica. FEMS Yeast Res. 2015;15:fov031 (Nielsen J, editor).View ArticleGoogle Scholar
- Blazeck J, Liu L, Redden H, Alper H. Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach. Appl Environ Microbiol. 2011;77:7905–14.View ArticleGoogle Scholar
- Martinez-Vazquez A, Gonzalez-Hernandez A, Domínguez Á, Rachubinski R, Riquelme M, Cuellar-Mata P, et al. Identification of the transcription factor Znc1p, which regulates the yeast-to-hypha transition in the dimorphic yeast Yarrowia lipolytica. PLoS ONE. 2013;8:e66790 (Bassilana M, editor).View ArticleGoogle Scholar
- Pédelacq J-D, Cabantous S, Tran T, Terwilliger TC, Waldo GS. Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol. 2006;24:79–88.View ArticleGoogle Scholar
- Fickers P, Le Dall M, Gaillardin C, Thonart P, Nicaud J. New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica. J Microbiol Methods. 2003;55:727–37.View ArticleGoogle Scholar
- Yang J, Nie L, Chen B, Liu Y, Kong Y, Wang H, et al. Hygromycin-resistance vectors for gene expression in Pichia pastoris. Yeast Chichester Engl. 2014;31:115–25.View ArticleGoogle Scholar
- Fennessy D, Grallert A, Krapp A, Cokoja A, Bridge AJ, Petersen J, et al. Extending the Schizosaccharomyces pombe molecular genetic toolbox. PLoS ONE. 2014;9:e97683.View ArticleGoogle Scholar
- Carroll Anne M, Sweigard James A. Barbara valent. Improved vectors for selecting resistance to hygromycin. Fungal Genet Newsl. 1994;41:22.Google Scholar
- Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, van den Hondel CA. Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene. 1987;56:117–24.View ArticleGoogle Scholar
- Le Dall MT, Nicaud JM, Gaillardin C. Multiple-copy integration in the yeast Yarrowia lipolytica. Curr Genet. 1994;26:38–44.View ArticleGoogle Scholar
- Dujon B, Sherman D, Fischer G, Durrens P, Casaregola S, Lafontaine I, et al. Genome evolution in yeasts. Nature. 2004;430:35–44.View ArticleGoogle Scholar
- Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 2009;6:343–5.View ArticleGoogle Scholar
- Fournier P, Abbas A, Chasles M, Kudla B, Ogrydziak DM, Yaver D, et al. Colocalization of centromeric and replicative functions on autonomously replicating sequences isolated from the yeast Yarrowia lipolytica. Proc Natl Acad Sci USA. 1993;90:4912–6.View ArticleGoogle Scholar
- Pan P, Hua Q. Reconstruction and in silico analysis of metabolic network for an oleaginous yeast, Yarrowia lipolytica. PLoS ONE. 2012;7:e51535.View ArticleGoogle Scholar
- Ratledge C, Wynn JP. The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv Appl Microbiol. 2002;51:1–51.View ArticleGoogle Scholar
- Dulermo T, Lazar Z, Dulermo R, Rakicka M, Haddouche R, Nicaud J-M. Analysis of ATP-citrate lyase and malic enzyme mutants of Yarrowia lipolytica points out the importance of mannitol metabolism in fatty acid synthesis. Biochim Biophys Acta. 2015;1851:1107–17.View ArticleGoogle Scholar
- Qiao K, Imam Abidi SH, Liu H, Zhang H, Chakraborty S, Watson N, et al. Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica. Metab Eng. 2015;29:56–65.View ArticleGoogle Scholar
- Tai M, Stephanopoulos G. Engineering the push and pull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production. Metab Eng. 2013;15:1–9.View ArticleGoogle Scholar
- Ivessa AS, Schneiter R, Kohlwein SD. Yeast acetyl-CoA carboxylase is associated with the cytoplasmic surface of the endoplasmic reticulum. Eur J Cell Biol. 1997;74:399–406.Google Scholar
- Hoja U, Marthol S, Hofmann J, Stegner S, Schulz R, Meier S, et al. HFA1 encoding an organelle-specific acetyl-CoA carboxylase controls mitochondrial fatty acid synthesis in Saccharomyces cerevisiae. J Biol Chem. 2004;279:21779–86.View ArticleGoogle Scholar
- Kolodziej SJ, Penczek PA, Schroeter JP, Stoops JK. Structure-function relationships of the Saccharomyces cerevisiae fatty acid synthase. Three-dimensional structure. J Biol Chem. 1996;271:28422–9.View ArticleGoogle Scholar
- Tehlivets O, Scheuringer K, Kohlwein SD. Fatty acid synthesis and elongation in yeast. Biochim Biophys Acta. 2007;1771:255–70.View ArticleGoogle Scholar
- Athenstaedt K, Jolivet P, Boulard C, Zivy M, Negroni L, Nicaud J-M, et al. Lipid particle composition of the yeast Yarrowia lipolytica depends on the carbon source. Proteomics. 2006;6:1450–9.View ArticleGoogle Scholar
- Athenstaedt K, Daum G. Biosynthesis of phosphatidic acid in lipid particles and endoplasmic reticulum of Saccharomyces cerevisiae. J Bacteriol. 1997;179:7611–6.View ArticleGoogle Scholar
- Benghezal M, Roubaty C, Veepuri V, Knudsen J, Conzelmann A. SLC1 and SLC4 encode partially redundant acyl-coenzyme A 1-acylglycerol-3-phosphate O-acyltransferases of budding yeast. J Biol Chem. 2007;282:30845–55.View ArticleGoogle Scholar
- Jain S, Stanford N, Bhagwat N, Seiler B, Costanzo M, Boone C, et al. Identification of a novel lysophospholipid acyltransferase in Saccharomyces cerevisiae. J Biol Chem. 2007;282:30562–9.View ArticleGoogle Scholar
- Ayciriex S, Le Guédard M, Camougrand N, Velours G, Schoene M, Leone S, et al. YPR139c/LOA1 encodes a novel lysophosphatidic acid acyltransferase associated with lipid droplets and involved in TAG homeostasis. Mol Biol Cell. 2012;23:233–46.View ArticleGoogle Scholar
- Henry SA, Kohlwein SD, Carman GM. Metabolism and regulation of glycerolipids in the yeast Saccharomyces cerevisiae. Genetics. 2012;190:317–49.View ArticleGoogle Scholar
- Kohlwein SD, Veenhuis M, van der Klei IJ. Lipid droplets and peroxisomes: key players in cellular lipid homeostasis or a matter of fat–store ‘em up or burn’em down. Genetics. 2013;193:1–50.View ArticleGoogle Scholar
- Carman GM, Han G-S. Regulation of phospholipid synthesis in the yeast Saccharomyces cerevisiae. Annu Rev Biochem. 2011;80:859–83.View ArticleGoogle Scholar
- Pascual F, Carman GM. Phosphatidate phosphatase, a key regulator of lipid homeostasis. Biochim Biophys Acta. 2013;1831:514–22.View ArticleGoogle Scholar
- Raetz CR. Enzymology, genetics, and regulation of membrane phospholipid synthesis in Escherichia coli. Microbiol Rev. 1978;42:614–59.Google Scholar
- Carman GM, Han G-S. Phosphatidic acid phosphatase, a key enzyme in the regulation of lipid synthesis. J Biol Chem. 2009;284:2593–7.View ArticleGoogle Scholar
- Su W-M, Han G-S, Casciano J, Carman GM. Protein kinase A-mediated phosphorylation of Pah1p phosphatidate phosphatase functions in conjunction with the Pho85p-Pho80p and Cdc28p-cyclin B kinases to regulate lipid synthesis in yeast. J Biol Chem. 2012;287:33364–76.View ArticleGoogle Scholar
- Su W-M, Han G-S, Carman GM. Cross-talk phosphorylations by protein kinase C and Pho85p-Pho80p protein kinase regulate Pah1p phosphatidate phosphatase abundance in Saccharomyces cerevisiae. J Biol Chem. 2014;289:18818–30.View ArticleGoogle Scholar
- Choi H-S, Su W-M, Han G-S, Plote D, Xu Z, Carman GM. Pho85p-Pho80p phosphorylation of yeast Pah1p phosphatidate phosphatase regulates its activity, location, abundance, and function in lipid metabolism. J Biol Chem. 2012;287:11290–301.View ArticleGoogle Scholar
- Athenstaedt K. YALI0E32769 g (DGA1) and YALI0E16797 g (LRO1) encode major triacylglycerol synthases of the oleaginous yeast Yarrowia lipolytica. Biochim Biophys Acta. 2011;1811:587–96.View ArticleGoogle Scholar
- Beopoulos A, Haddouche R, Kabran P, Dulermo T, Chardot T, Nicaud J-M. Identification and characterization of DGA2, an acyltransferase of the DGAT1 acyl-CoA:diacylglycerol acyltransferase family in the oleaginous yeast Yarrowia lipolytica. New insights into the storage lipid metabolism of oleaginous yeasts. Appl Microbiol Biotechnol. 2012;93:1523–37.View ArticleGoogle Scholar
- Oelkers P, Tinkelenberg A, Erdeniz N, Cromley D, Billheimer JT, Sturley SL. A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast. J Biol Chem. 2000;275:15609–12.View ArticleGoogle Scholar
- Zweytick D, Leitner E, Kohlwein SD, Yu C, Rothblatt J, Daum G. Contribution of Are1p and Are2p to steryl ester synthesis in the yeast Saccharomyces cerevisiae. Eur J Biochem FEBS. 2000;267:1075–82.View ArticleGoogle Scholar
- Dulermo T, Tréton B, Beopoulos A, Kabran Gnankon AP, Haddouche R, Nicaud J-M. Characterization of the two intracellular lipases of Y. lipolytica encoded by TGL3 and TGL4 genes: new insights into the role of intracellular lipases and lipid body organisation. Biochim Biophys Acta. 2013;1831:1486–95.View ArticleGoogle Scholar
- Ham HJ, Rho HJ, Shin SK, Yoon H-J. The TGL2 gene of Saccharomyces cerevisiae encodes an active acylglycerol lipase located in the mitochondria. J Biol Chem. 2010;285:3005–13.View ArticleGoogle Scholar
- Ansari AM, Ahmed AK, Matsangos AE, Lay F, Born LJ, Marti G, et al. Cellular GFP toxicity and immunogenicity: potential confounders in in vivo cell tracking experiments. Stem Cell Rev Rep. 2016;12:553–9.View ArticleGoogle Scholar
- Breker M, Gymrek M, Moldavski O, Schuldiner M. LoQAtE–localization and quantitation ATlas of the yeast proteomE. A new tool for multiparametric dissection of single-protein behavior in response to biological perturbations in yeast. Nucleic Acids Res. 2014;42:D726–30.View ArticleGoogle Scholar
- Huh W-K, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS, et al. Global analysis of protein localization in budding yeast. Nature. 2003;425:686–91.View ArticleGoogle Scholar
- Jonikas MC, Collins SR, Denic V, Oh E, Quan EM, Schmid V, et al. Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum. Science. 2009;323:1693–7.View ArticleGoogle Scholar
- Dean N, Zhang YB, Poster JB. The VRG4 gene is required for GDP-mannose transport into the lumen of the Golgi in the yeast, Saccharomyces cerevisiae. J Biol Chem. 1997;272:31908–14.View ArticleGoogle Scholar
- Hung N-J, Johnson AW. Nuclear recycling of the Pre-60S ribosomal subunit-associated factor Arx1 depends on Rei1 in Saccharomyces cerevisiae. Mol Cell Biol. 2006;26:3718–27.View ArticleGoogle Scholar
- Hung N-J, Lo K-Y, Patel SS, Helmke K, Johnson AW. Arx1 is a nuclear export receptor for the 60S ribosomal subunit in yeast. Mol Biol Cell. 2008;19:735–44.View ArticleGoogle Scholar
- Foster MW, Forrester MT, Stamler JS. A protein microarray-based analysis of S-nitrosylation. Proc Natl Acad Sci USA. 2009;106:18948–53.View ArticleGoogle Scholar
- Orozco H, Matallana E, Aranda A. Oxidative stress tolerance, adenylate cyclase, and autophagy are key players in the chronological life span of Saccharomyces cerevisiae during Winemaking. Appl Environ Microbiol. 2012;78:2748–57.View ArticleGoogle Scholar
- Reinders J, Zahedi RP, Pfanner N, Meisinger C, Sickmann A. Toward the complete yeast mitochondrial proteome: multidimensional separation techniques for mitochondrial proteomics. J Proteome Res. 2006;5:1543–54.View ArticleGoogle Scholar
- Hess DC, Myers CL, Huttenhower C, Hibbs MA, Hayes AP, Paw J, et al. Computationally driven, quantitative experiments discover genes required for mitochondrial biogenesis. PLoS Genet. 2009;5:e1000407 (Kim SK, editor).View ArticleGoogle Scholar
- Delmer DP. Dimethylsulfoxide as a potential tool for analysis of compartmentation in living plant cells. Plant Physiol. 1979;64:623–9.View ArticleGoogle Scholar
- Evans Christopher T, Ratledge Colin. Effect of nitrogen source on lipid accumulation in Oleaginous yeasts. J Gen Microbiol. 1984;130:1693–704.Google Scholar
- Park WS, Murphy PA, Glatz BA. Lipid metabolism and cell composition of the oleaginous yeast Apiotrichum curvatum grown at different carbon to nitrogen ratios. Can J Microbiol. 1990;36:318–26.View ArticleGoogle Scholar
- Cescut J, Fillaudeau L, Molina-Jouve C, Uribelarrea J-L. Carbon accumulation in Rhodotorula glutinis induced by nitrogen limitation. Biotechnol Biofuels. 2014;7:164.View ArticleGoogle Scholar
- Calvey CH, Su Y-K, Willis LB, McGee M, Jeffries TW. Nitrogen limitation, oxygen limitation, and lipid accumulation in Lipomyces starkeyi. Bioresour Technol. 2016;200:780–8.View ArticleGoogle Scholar
- Liu T, Li Y, Liu F, Wang C. The enhanced lipid accumulation in oleaginous microalga by the potential continuous nitrogen-limitation (CNL) strategy. Bioresour Technol. 2016;203:150–9.View ArticleGoogle Scholar
- Ushinsky SC, Bussey H, Ahmed AA, Wang Y, Friesen J, Williams BA, et al. Histone H1 in Saccharomyces cerevisiae. Yeast Chichester Engl. 1997;13:151–61.View ArticleGoogle Scholar
- Sorger D, Athenstaedt K, Hrastnik C, Daum G. A yeast strain lacking lipid particles bears a defect in ergosterol formation. J Biol Chem. 2004;279:31190–6.View ArticleGoogle Scholar
- Chung S-K, Lee K-W, Kang HI, Yamashita C, Kudo M, Yoshida Y. Design and synthesis of potential inhibitors of the ergosterol biosynthesis as antifungal agents. Bioorg Med Chem. 2000;8:2475–86.View ArticleGoogle Scholar
- Van Den Hazel HB, Kielland-Brandt MC, Winther JR. Review: biosynthesis and function of yeast vacuolar proteases. Yeast Chichester Engl. 1996;12:1–16.View ArticleGoogle Scholar
- Spencer J, Phister TG, Smart KA, Greetham D. Tolerance of pentose utilising yeast to hydrogen peroxide-induced oxidative stress. BMC Res. Notes. 2014;7:151.View ArticleGoogle Scholar
- Lopes M, Mota M, Belo I. Comparison of Yarrowia lipolytica and Pichia pastoris cellular response to different agents of oxidative stress. Appl Biochem Biotechnol. 2013;170:448–58.View ArticleGoogle Scholar
- Biryukova EN, Medentsev AG, Arinbasarova AY, Akimenko VK. Tolerance of the yeast Yarrowia lipolytica to oxidative stress. Microbiology. 2006;75:243–7.View ArticleGoogle Scholar
- Kawasse FM, Amaral PF, Rocha-Leão MHM, Amaral AL, Ferreira EC, Coelho MAZ. Morphological analysis of Yarrowia lipolytica under stress conditions through image processing. Bioprocess Biosyst Eng. 2003;25:371–5.View ArticleGoogle Scholar
- Eitzen GA, Szilard RK, Rachubinski RA. Enlarged peroxisomes are present in oleic acid-grown Yarrowia lipolytica overexpressing the PEX16 gene encoding an intraperoxisomal peripheral membrane peroxin. J Cell Biol. 1997;137:1265–78.View ArticleGoogle Scholar
- Szilard RK, Titorenko VI, Veenhuis M, Rachubinski RA. Pay32p of the yeast Yarrowia lipolytica is an intraperoxisomal component of the matrix protein translocation machinery. J Cell Biol. 1995;131:1453–69.View ArticleGoogle Scholar
- Titorenko VI, Smith JJ, Szilard RK, Rachubinski RA. Pex20p of the Yeast Yarrowia lipolytica is required for the oligomerization of thiolase in the cytosol and for its targeting to the peroxisome. J Cell Biol. 1998;142:403–20.View ArticleGoogle Scholar
- Wriessnegger T, Gübitz G, Leitner E, Ingolic E, Cregg J, de la Cruz BJ, et al. Lipid composition of peroxisomes from the yeast Pichia pastoris grown on different carbon sources. Biochim Biophys Acta. 2007;1771:455–61.View ArticleGoogle Scholar
- Nazarko T, Nicaud J, Sibirny A. Observation of the peroxisome-vacuole dynamics by fluorescence microscopy with a single filter set. Cell Biol Int. 2005;29:65–70.View ArticleGoogle Scholar
- Chang J, Mast FD, Fagarasanu A, Rachubinski DA, Eitzen GA, Dacks JB, et al. Pex3 peroxisome biogenesis proteins function in peroxisome inheritance as class V myosin receptors. J Cell Biol. 2009;187:233–46.View ArticleGoogle Scholar
- Kabran P, Rossignol T, Gaillardin C, Nicaud J-M, Neuveglise C. Alternative splicing regulates targeting of malate dehydrogenase in Yarrowia lipolytica. DNA Res. 2012;19:231–44.View ArticleGoogle Scholar
- Dulermo T, Nicaud J-M. Involvement of the G3P shuttle and β-oxidation pathway in the control of TAG synthesis and lipid accumulation in Yarrowia lipolytica. Metab Eng. 2011;13:482–91.View ArticleGoogle Scholar
- Gould SJ, Kalish JE, Morrell JC, Bjorkman J, Urquhart AJ, Crane DI. Pex13p is an SH3 protein of the peroxisome membrane and a docking factor for the predominantly cytoplasmic PTs1 receptor. J Cell Biol. 1996;135:85–95.View ArticleGoogle Scholar
- Elgersma Y, Kwast L, Klein A, Voorn-Brouwer T, van den Berg M, Metzig B, et al. The SH3 domain of the Saccharomyces cerevisiae peroxisomal membrane protein Pex13p functions as a docking site for Pex5p, a mobile receptor for the import PTS1-containing proteins. J Cell Biol. 1996;135:97–109.View ArticleGoogle Scholar
- Smith JJ, Aitchison JD. Peroxisomes take shape. Nat Rev Mol Cell Biol. 2013;14:803–17.View ArticleGoogle Scholar
- Manjithaya R, Nazarko TY, Farré J-C, Subramani S. Molecular mechanism and physiological role of pexophagy. FEBS Lett. 2010;584:1367–73.View ArticleGoogle Scholar
- Hutchins MU, Veenhuis M, Klionsky DJ. Peroxisome degradation in Saccharomyces cerevisiae is dependent on machinery of macroautophagy and the Cvt pathway. J Cell Sci. 1999;112(Pt 22):4079–87.Google Scholar
- Cordero Otero R, Gaillardin C. Efficient selection of hygromycin-B-resistant Yarrowia lipolytica transformants. Appl Microbiol Biotechnol. 1996;46:143–8.View ArticleGoogle Scholar
- Ruiz-Pavón L, Domínguez A. Characterization of the Yarrowia lipolytica YlSRP72 gene, a component of the yeast signal recognition particle. Int Microbiol Off J Span Soc Microbiol. 2007;10:283–9.Google Scholar
- Handee W, Li X, Hall KW, Deng X, Li P, Benning C, et al. An energy-independent pro-longevity function of triacylglycerol in yeast. PLOS Genet. 2016;12:e1005878 (Longo VD, editor).View ArticleGoogle Scholar
- Pomraning KR, Kim Y-M, Nicora CD, Chu RK, Bredeweg EL, Purvine SO, et al. Multi-omics analysis reveals regulators of the response to nitrogen limitation in Yarrowia lipolytica. BMC Genomics [Internet]. 2016; 17. http://www.biomedcentral.com/1471-2164/17/138. Accessed 30 Mar 2016.
- Mekouar M, Blanc-Lenfle I, Ozanne C, Da Silva C, Cruaud C, Wincker P, et al. Detection and analysis of alternative splicing in Yarrowia lipolytica reveal structural constraints facilitating nonsense-mediated decay of intron-retaining transcripts. Genome Biol. 2010;11:R65.View ArticleGoogle Scholar
- Morin N, Cescut J, Beopoulos A, Lelandais G, Le Berre V, Uribelarrea J-L, et al. Transcriptomic analyses during the transition from biomass production to lipid accumulation in the oleaginous yeast Yarrowia lipolytica. PLoS ONE. 2011;6:e27966.View ArticleGoogle Scholar
- Colot HV, Park G, Turner GE, Ringelberg C, Crew CM, Litvinkova L, et al. A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. Proc Natl Acad Sci. 2006;103:10352–7.View ArticleGoogle Scholar
- Jefferson RA, Kavanagh TA, Bevan MW. GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 1987;6:3901–7.Google Scholar
- Holsters M, de Waele D, Depicker A, Messens E, van Montagu M, Schell J. Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet MGG. 1978;163:181–7.View ArticleGoogle Scholar
- de Groot MJA, Bundock P, Hooykaas PJ, Beijersbergen AGM. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol. 1998;16:839–42.View ArticleGoogle Scholar
- Murray MG, Thompson WF. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 1980;8:4321–6.View ArticleGoogle Scholar
- Zerbino DR. Using the Velvet de novo assembler for short-read sequencing technologies. Curr Protoc Bioinforma. Ed. Board Andreas Baxevanis Al. 2010; Chapter 11: Unit 11.5.
- Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinforma Oxf Engl. 2009;25:1105–11.View ArticleGoogle Scholar
- Götz S, García-Gómez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, et al. High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res. 2008;36:3420–35.View ArticleGoogle Scholar
- Kent WJ. BLAT–the BLAST-like alignment tool. Genome Res. 2002;12:656–64.View ArticleGoogle Scholar
- Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10:R25.View ArticleGoogle Scholar
- Gritz L, Davies J. Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene. 1983;25:179–88.View ArticleGoogle Scholar
- Gietz RD, Woods RA. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 2002;350:87–96.View ArticleGoogle Scholar
- Ghaemmaghami S, Huh W-K, Bower K, Howson RW, Belle A, Dephoure N, et al. Global analysis of protein expression in yeast. Nature. 2003;425:737–41.View ArticleGoogle Scholar
- Gietz RD. Yeast transformation by the LiAc/SS carrier DNA/PEG method. Methods Mol Biol Clifton NJ. 2014;1163:33–44.View ArticleGoogle Scholar
- Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671–5.View ArticleGoogle Scholar
- Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, et al. Cell profiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 2006;7:R100.View ArticleGoogle Scholar
- Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9:676–82.View ArticleGoogle Scholar
- Gaillardin CM, Charoy V, Heslot H. A study of copulation, sporulation and meiotic segregation in Candida lipolytica. Arch Mikrobiol. 1973;92(1):69–83.View ArticleGoogle Scholar