Construction of isogenic NHEJ-deficient auxotrophic strains

Previous work has shown that a deletion of the ku70 ortholog increases transformation efficiency and rate of recovery of transformants targeted to specific loci [11, 12]. This removes a non-homologous DNA repair process which allows random integration of DNA and so decreases mis-localization of constructs intended for a particular locus. The Y. lipolytica ku70 ortholog was identified as YALI0C08701g by BLAST. We set out to construct a set of isogenic strains in which ku70 was replaced with a gene conferring hygromycin resistance (hygromycin phosphotransferase; hph) in the Po1g genetic background, commonly used for studies in fungal biotechnology [7, 30] (Table 1). The majority of selective markers available in Y. lipolytica are amino acid and nucleobase auxotrophies. Hygromycin B, derived from Streptomyces hygroscopicus, is a translation inhibitor effective in other fungal systems. It has been demonstrated to be useful in Y. lipolytica in a loxP Cre-removable system [11, 38]. Hygromycin B is an archival and successful selective agent for ascomycetes and hemiascomycetes [39–42]. The wild-type strains ATCC20460™ and ATCC18944 ™ as well as Po1g have different natural resistance levels to the antibiotic Hygromycin B (see Additional file 1: Figure S1). Efficient expression of hph is key for use as a selective marker. We therefore codon optimized hph to enable stricter selection. In order to produce auxotrophic markers leu2 and ura3 separately and together, we replaced ku70 (YALI0C08701g) with hph driven by the tef promoter in the leu2 auxotroph Po1g as a starting point to make FKP355 (matA, ku70::hph+, leu2-270) (Fig. 1b). Successful replacement was confirmed by Southern blot. Figure S2E (see Additional file 1) shows the proper length of all genomic DNA fragments after digestion with BglII, EcoRV, or PvuII restriction endonucleases. We then complemented leu2-270 by replacement with leu2+ to make FKP391 (matA, ku70::hph+, leu2-270::leu2+), providing a prototroph. To generate a double auxotroph from leucine-requiring FKP355, we selected for disruption of ura3 by selection on 5-FOA containing medium to make FKP393 (matA, ku70::hph+, leu2-270, ura3 ⁻). The complicated ura3 locus in FKP355 contains ura3::suc2+ adjacent to an intact copy of ura3. The disrupted and intact copies of ura3 recombined leaving only ura3::suc2+ in FKP393. Analysis of RNA suggests that the plasmid sequence between the disrupted and intact copies of ura3 was lost, indicative of a loop out event while genes adjacent to ura3 (YALI0E26653g and YALI0E26785g) are intact (RNA data not shown). We then complemented leu2-270 in FKP393 by replacement with leu2+ to make FEB130 (matA, ku70::hph+, ura3 ⁻) using the gene from wild-type strain W29 (ATCC 20460™) [9]. These strains represent a complimented prototroph and a set of auxotrophs in an isogenic NHEJ-deficient background for biological investigation (Fig. 1b).

Strain	Genotype	Reference
W29	ATCC20460	Gaillardin et al. [137]
P01g	matA, xpr2-332, axp-2, leu2-270	Madzak et al. [7]
FKP355	matA, xpr2-332, axp-2, ku70::hph+, leu2-270	This work
FKP391	matA, xpr2-332, axp-2, ku70::hph+, leu2-270::leu2+	This work
FKP393	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, ura3	This work
FEB130	matA, xpr2-332, axp-2, ku70::hph+, leu2-270::leu2+, ura3	This work
FEB56	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, erg6-sfGFP:leu2+	This work
FEB64	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, pex13-sfGFP:leu2+	This work
FEB91	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, hH1-sfGFP:leu2+	This work
FEB92	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, rdl2-sfGFP:leu2+	This work
FEB93	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, aim17-sfGFP:leu2+	This work
FEB94	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, emc2-sfGFP:leu2+	This work
FEB96	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, cpy1-sfGFP:leu2+	This work
FEB97	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, arx1-sfGFP:leu2+	This work
FEB103	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, vrg4-sfGFP:leu2+	This work
FEB100	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, erg6-sfGFP:leu2+, ura3	This work
FEB98	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, pex13-sfGFP:leu2+, ura3	This work
FEB87	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, hH1-sfGFP:leu2+, ura3	This work
FEB83	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, rdl2-sfGFP:leu2+, ura3	This work
FEB84	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, aim17-sfGFP:leu2+, ura3	This work
FEB89	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, emc2-sfGFP:leu2+, ura3	This work
FEB85	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, cpy1-sfGFP:leu2+, ura3	This work
FEB86	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, arx1-sfGFP:leu2+, ura3	This work
FEB90	matA, xpr2-332, axp-2, ku70::hph+, leu2-270, vrg4-sfGFP:leu2+, ura3	This work

Genome sequencing, assembly, and annotation of NHEJ-deficient base strain

Construction of a multipurpose vector for rapid expression of fluorescently tagged proteins in Yarrowia lipolytica

We constructed a multipurpose expression vector pYL15 (GenBank accession: KU378202) containing a codon optimized version of the superfolder GFP gene (sfGFP) [37] preceded by a 10× glycine linker and driven by the high expression exp1 promoter [35] to enable rapid analysis of protein localization by expression of GFP fusion proteins in Y. lipolytica. Coding sequences of interest are amplified by PCR from genomic or complementary DNA with primers containing short overhangs homologous to either side of a SmaI site present in the pYL15 vector, which contains the full length leu2 gene from Y. lipolytica. The vector and coding sequence are then assembled by Gibson assembly [45] to reduce the chances of producing vector without an insert (Fig. 2). The vector contains centromere and origin of replication sequences from ARS68 [46] enabling autonomous replication when transformed as a circular plasmid but when linear integrates at the leu2-270 locus present in strains commonly used for Y. lipolytica genetic studies [7, 43] (Fig. 2). Transformed cells are typically grown in yeast nitrogen base medium with ammonium sulfate (YNB) requiring biosynthesis of leucine to select for maintenance of the plasmid. This system enables rapid assembly and high-level expression of sfGFP-tagged proteins in Y. lipolytica. The ability to clone any coding sequence without the use of restriction enzymes and using a single linear plasmid makes this construct amenable to moderate- and high-throughput protein localization studies. To demonstrate the usefulness of pYL15, we cloned enzymes involved in triglyceride biosynthesis and expressed them extra-chromosomally with sfGFP tags to determine their localization.

Expression of sfGFP-tagged lipid biosynthetic enzymes reveals their localization

Yarrowia lipolytica is a model organism for the study of metabolism in the context of lipid production for biofuels and other value added lipid products. A number of compartmentalized metabolic models have been constructed for Y. lipolytica that incorporate data derived primarily from homologous proteins in other organisms [15, 16, 47]. We therefore assessed the localization of enzymes involved in triglyceride biosynthesis from glucose in Y. lipolytica to determine where key metabolic reactions occur and to improve on location-dependent metabolic models in Y. lipolytica (Fig. 3; Table 3). The initial building block for fatty acids is Acetyl-CoA, which is produced from citrate by ATP-ctirate lyase (ACL). The presence of ACL in Y. lipolytica is a major distinction between its metabolic network and that of non-oleaginous organisms [48] and deletion results in reduced ability to accumulate lipids [49]. Both subunits of this enzyme localize to the cytosol where they presumably interact to drive lipid accumulation from citrate. Conversion of Acetyl-CoA to Malonyl-CoA by Acetyl-CoA Carboxylase (ACC) is the committed step in fatty acid production and has been successfully used to produce higher quantities of lipids in Y. lipolytica [50, 51]. In Saccharomyces cerevisiae, ACC is expressed by two genes, acc1 and hfa1, which encode cytosol- [52], and mitochondria-localized [53] variants. Y. lipolytica has a single copy of acc1 with a putative mitochondrial targeting signal that exhibits punctate localization that partially overlaps with mitochondria (Figs. 3, 4). Acetyl-CoA and Malonyl-CoA are then utilized by the Type I Fatty Acid Synthase complex, which in S. cerevisiae consists of six copies each of cytoplasmically located Fas1p and Fas2p [54, 55] and Beta-ketoacyl-ACP synthase to make acyl-CoA molecules which primarily consist of 16:0, 18:1, 18:2 chains when grown on glucose [28, 56]. The genes encoding homologs of fas1 and fas2 in Y. lipolytica are both predicted to have short introns. Intriguingly, the splice junction at the 5′ end of the intron is within the translation start site of fas2 and directly adjacent to the translation start site of fas1, respectively, potentially enabling splicing as a strong regulator of expression for these genes. We were unable to detect a green fluorescent protein signal in fas1-sfGFP or fas2-sfGFP strains constructed with or without the short leader introns (data not shown) suggesting that translation of fas1 and fas2 may be tightly controlled by an as yet unknown mechanism in Y. lipolytica. Acyl-CoA molecules produced by the fatty acid synthase complex are utilized by enzymes during lipid biosynthesis. The initial acyl group is attached to the lipid backbone by the glycerol-3-phosphate sn-1 acyltransferase sct1, which we were unable to detect green fluorescent protein signal from. A second acyl chain can be added to lyso-phosphatidic acid by a number of different enzymes in S. cerevisiae including Slc1p [57], Ale1p [58, 59], and Loa1p [60], to produce phosphatidic acid. Interestingly, we found that Slc1-sfGFPp localizes to both the endoplasmic reticulum and the periphery of lipid droplets while Ale1-sfGFPp and Loa1-sfGFPp localize exclusively to the endoplasmic reticulum, consistent with results in S. cerevisiae [61, 62] (Figs. 3, 5). Utilization of phosphatidic acid represents a major regulatory point in lipid homeostasis (for review see: [62–64]). CDP-Diacylglycerol Synthase (CDS) activates phosphatidic acid by addition of CDP prior to release of CMP during the construction of the polar head groups for phospholipid synthesis [65], localized in the ER in Y. lipolytica (Fig. 3). Alternatively, phosphatidic acid is dephosphorylated to form diacylglycerol by Phosphatidate Phosphatase. In S. cerevisiae, the Phosphatidate Phosphatase Pah1p is regulated by phosphorylation to maintain lipid homeostasis between storage and phospholipids [66–69], and the homolog of Pah1p in Y. lipolytica resides in the cytosol (Fig. 3). The endoplasmic reticulum localized enzyme Diacylglycerol Kinase (Dgk1-sfGFPp) phosphorylates diacylglycerol to produce phosphatidic acid (Fig. 3). Alternately, acylation of diacylglycerol to produce storage lipids in the form of triglycerides is performed by Dga1p, Dga2p, and Lro1p. Dga1p and Dga2p utilize acyl-CoA as the acyl chain donor while the lecithin cholesterol acyltransferase Lro1p utilizes phospholipids [70–72]. Lro1-sfGFPp localizes to the endoplasmic reticulum and like Loa1-sfGFPp and Dgk1-sfGFPp is found primarily at the nuclear periphery (Fig. 3). In contrast, Dga1-sfGFPp localizes to the endoplasmic reticulum and the periphery of lipid droplets (Figs. 3, 5), suggesting it is the major producer of lipid droplet stored triglycerides in Y. lipolytica, while Dga2-sfGFPp localizes exclusively to the endoplasmic reticulum (Fig. 3). Acyl-CoA is also used for the esterification of sterols by Are1p [73] which localizes to the endoplasmic reticulum in Y. lipolytica (Fig. 3). We are also interested in the localization of enzymes that utilize triglycerides. Y. lipolytica has four triglyceride lipases that catalyze the cleavage of triglycerides to produce free fatty acids. We found that three of them localize to lipid droplets (Tgl1-sfGFPp, Tgl3-sfGFPp, and Tgl4-sfGFPp; Fig. 5) in a manner consistent with previous findings for Tgl3p and Tgl4p [74], while Tgl2-sfGFPp localizes to the mitochondria (Figs. 3, 4) as in S. cerevisiae [75].

Y. lipolytica gene	S. cerevisiae homolog	sfGFP tag	S. cerevisiae
YALI0C11407g (acc1)	acc1, hfa1	MI	CY, MI
YALI0E34793g (acl1)	–	CY	–
YALI0D24431g (acl2)	–	CY	–
YALI0F19514g (ale1)	ale1	ER	ER
YALI0F06578g (are1)	are1	ER	ER
YALI0D07986g (dga2)	are2	ER	ER
YALI0E14443g (cds1)	cds1	ER	ER, MI
YALI0E32769g (dga1)	dga1	ER, LD	ER, LD
YALI0F19052g (dgk1)	dgk1	ER	ER
YALI0C14014g (loa1)	loa1	ER	ER, LD
YALI0E16797g (lro1)	lro1	ER	ER
YALI0D27016g (pah1)	pah1	CY	CY, ER
YALI0E18964g (slc1)	slc1	ER, LD	ER, LD
YALI0E32035g (tgl1)	tgl1	LD	ER, LD
YALI0E31515g (tgl2)	tgl2	MI	MI
YALI0D17534g (tgl3)	tgl3	LD	LD
YALI0F10010g (tgl4)	tgl4	LD	LD

The locations we determined are frequently consistent with homologs of enzymes in S. cerevisiae [61, 62] with all five of the exceptions being enzymes where we identified only a single location in Y. lipolytica but for which multiple locations have been determined in S. cerevisiae (Fig. 3). Additional experimentation in Y. lipolytica is likely to augment these additional locations. For example, we determined that Pah1-sfGFPp is localized to the cytosol; however, its substrate, phosphatidic acid, resides within the endoplasmic reticulum membrane suggesting that Pah1p must interact at least transiently with the endoplasmic reticulum. We found that many of the lipid biosynthesis enzymes localize to the endoplasmic reticulum; however, GFP data alone cannot determine ER lumen or cytosol direction of enzyme activity. In summary, we identified the location of 17 enzymes involved in lipid metabolism in Y. lipolytica by plasmid-based expression, many of which localize to multiple organelles (Fig. 6). These data provide evidence for additional locations of Dga1p and Slc1p and previously undetermined locations for Dga2p and Loa1p that can be used to improve compartmentalized metabolic models for Y. lipolytica [15].

Construction of an atlas of endogenous sfGFP-tagged organelles

To overcome limitations of organelle identification by staining and fixation, we generated an atlas of strains with green fluorescent organelles by tagging genes with sfGFP at their endogenous locus (Figs. 7, 8). These intracellular markers respond to native promoter control, rather than being constitutively expressed from plasmids, and avoid overexpression toxicity, which has been suggested in other eukaryotic systems [76]. We chose non-essential proteins which show high and consistent expression, membrane association to define the organelle, DNA or rRNA association for the nucleus, and successful tagging in the S. cerevisiae GFP protein libraries [77, 78]. We identified genes encoding Y. lipolytica homologs of proteins known to be specific to particular organelles in S. cerevisiae including proteins that localize to the nucleus (hH1, YALI0B16280g; arx1, YALI0C05599g), mitochondrion (rdl2, YALI0F29667g; aim17, YALI0F16357g), peroxisome (pex13, YALI0C05775g), lipid droplet (erg6, YALI0F08701g), endoplasmic reticulum (emc2, YALI023188g), vacuole (cpy1, YALI0A18810g), and golgi apparatus (vrg4, YALI0F21791g) (Table 4). We constructed the green fluorescent organelle atlas in a prototrophic and auxotrophic (ura3−) ku70::hph genetic background to enable observation of experimental perturbations in mutant and wild-type–sfGFP strains, and to provide a convenient tool for co-localization studies.

Name	Y. lipolytica	S.cerevisiae	Coverage (%)	Identity (%)
hH1	YALI0B16280g	YPL127C	59	54
arx1	YALI0C05599g	YDR101C	90	33
cpy/prc1	YALI0A18810g	YMR297W	83	66
vrg4	YALI0F21791p	YGL225W	94	61
aim17	YALI0F16357p	YHL021C	89	28
rdl2	YALI0F29667g	YOR286W	67	49
emc2	YALI0C23188g	YJR088C	92	25
pex13	YALI0C05775g	YLR191W	79	50
erg6	YALI0F08701g	YML008C	94	65

For all markers tested, localization in Y. lipolytica is consistent with S. cerevisiae orthologs and previous work [77, 78]. The function of the endoplasmic reticulum marker Emc2p is unrelated to lipid biogenesis, to avoid bias to a process of primary interest in Y. lipolytica. Emc2p is a member of a transmembrane complex; when members are deleted or perturbed, S. cerevisiae cells show signs of the unfolded protein response [79]. Vrg4p is a golgi GDP-mannose transporter needed for protein glycosylation [80]. We tagged two proteins predicted to localize to the nucleus. The linker histone H1 directly interacts with DNA throughout the interior of the nucleus while the ribosomal export protein Arx1p shuttles the pre-60S subunit for export, and interacts with a number of nucleoporin subunits [81, 82]. The primary localization of Arx1p-sfGFP is also nuclear, at times sub-localized, presumably at the nucleolus (Figs. 7, 8). We tagged two mitochondrial proteins. Rdl2p is a rhodanese-like protein which has thiosulfate sulfurtransferase activity [83]. Rdl2p function in S. cerevisiae is linked to H₂O₂ detoxification, which contributes to life span in longer-lived wine fermentation yeast strains, where this protein is highly expressed in the late stages of fermentation [84]. Aim17p is implicated in mitochondrial biogenesis in S. cerevisiae [85, 86]. Additionally, we report specific and active localization through the course of experiments to characterize these proteins with existing tools and techniques. Specific localization of the mitochondrial markers Rdl2p and Aim17p was more discrete than the commercially available MitoTracker (Fig. 9), which contains a reactive thiol and is delivered in dimethylsulfoxide to perforate cell membranes [87]. Nitrogen limitation in a carbon-replete environment is a condition used to stimulate lipid accumulation in microalgal, algal, cyanobacterial, and fungal organisms, including Y. lipolytica, for biofuel development [88–92]. We evaluated the atlas proteins for expression and organelle-specific localization in YPD and lower nitrogen (Y-D, YPD lacking peptone) media in both our prototroph and auxotroph strains (Figs. 7, 8). Localization not only was consistent with the expected organelle compartments but also reflects responses to different nutrient types. We found several interesting patterns after transfer to nitrogen-deficient (Y-D) medium that may be associated with stationary phase such as histone H1, important for DNA packaging [93]. Erg6 is a protein in the ergosterol biosynthetic pathway, used as a lipid droplet marker, and a target of antifungals [94, 95]. It has also been identified by proteomics in Y. lipolytica lipid droplets [56], and here shows changing lipid droplet size and number. Cyp1p, a nonspecific carboxypeptidase involved in protein degradation [96], also exhibits altered vacuole size and number consistent with a role in degradation. In total, we have tagged proteins covering seven cellular compartments.

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 [97], a susceptibility also confirmed in Y. lipolytica [98]. Further, it has been noted that prior exposure of yeast to an oxidative stress increases tolerance to higher concentrations of ROS (reactive oxygen species) [97] suggesting a physiological response which may be based in protein expression. ROS tolerance is also increased in stationary vs. exponential phase cells [99]. 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.

To determine the response of organelle tags involved in detoxification of ROS, we grew cells expressing Rdl2-sfGFPp, Aim17-sfGFPp, and Pex13-sfGFPp to exponential and stationary phases. They were then treated with hydrogen peroxide and imaged immediately and again after 24 h. We observed a difference in growth phase susceptibility, and that Rdl2-sfGFPp signal intensity changes after H₂O₂ treatment (Fig. 10). Stationary phase cells have emblematic mitochondrial signal, which is distributed as seen in other growth conditions and stains (Figs. 7, 8, 9) after 24 h of stress. Exponential phase cells are somewhat elongated in all samples, indicative of stress [100], and have punctate mitochondrial and peroxisomal protein signals. These results suggest that Rdl2-sfGFPp distribution, such as between exponential and stationary phase cells (Fig. 10), has the potential to be used as a relative marker of oxidative stress. Consistent with Rdl2 expression levels correlating with stress tolerance in S. cerevisiae [84], monitored expression of Rdl2 under controlled and experimental growth conditions will aid optimization of bioproduct production in this obligate aerobe.

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 [104]. 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) [106]. 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 [107]. 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 [13]. Loss of peroxisome-localized beta-oxidation enzyme function resulting in increased lipid accumulation shows the importance of this organelle to biofuel development [108].

Pex13p in S. cerevisiae is a protein with a C-terminal SH3 domain used for docking peroxisomal targeting signal (PTS)-containing proteins, which are chaperones for peroxisome bound cargo [109, 110]. To demonstrate peroxisome labeling, Pex13-sfGFPp was grown with oleate as the sole carbon source (Fig. 11), which induces peroxisome formation. Pex13-sfGFPp signal is generally punctate but disperses somewhat after nitrogen limitation in the auxotrophic and prototrophic atlas strains (Figs. 7, 8). This suggests that Pex13p, an essential part of the protein import complex of Pex13/14/17 for movement into the peroxisomal matrix [111], may be redistributed or broken down in different media [105, 112]. To test this, we grew the Pex13-sfGFPp strain in rich medium (YPD) and then transferred it to rich and minimal (YNB) medium with and without (YNBnoN or Y-D) a nitrogen source for 16 h before microscopy. In both media types with reduced nitrogen, Pex13-sfGFPp signal is increased. Peroxisome signal is lost in minimal medium (YNB), and is greatest in nitrogen-deficient rich medium (Y-D) (Fig. 12). The signal quantification is in contrast to S. cerevisiae, which experiences induction of pexophagy by transfer from oleate media into glucose with nitrogen [113]. However, it is difficult to interpret total signal, as this could indicate redistribution or breakdown of the organelles. These data are evidence of different peroxisome behaviors in oleaginous organisms as compared to conventional yeast models. This strain set will allow minimization of peroxisome production and activity specific to Y. lipolytica without major biochemical pathway loss in the mutation of mfe1p.

Chemicals

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 [7] 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 [120] 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 [121], 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.

Agrobacterium-mediated transformation

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 [122]. 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 [123] 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 [124] 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 [125] 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 [126] to predict transcripts from which coding regions were predicted. Predicted proteins were functionally annotated using Blast2GO [127]. Full length transcripts were aligned to contigs from strain CLIB122 [44] using Blat v32×1 [128] to determine their position and percent identity. Reads were aligned to the reference genome using Bowtie v0.12.9 [129] and SNPs identified with custom Perl scripts.

Plasmid construction

A codon optimized superfolder GFP (sfGFP) gene [37] followed by a codon optimized hph gene from Escherichia coli [130] driven by the high expression Yarrowia lipolytica tef promoter along with the centromere and origin of replication sequences from ARS68 [46] 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 [131] 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 [133]. 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).

Microscopy

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 [134] and CellProfiler [135]. 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.