Overexpression screen reveals transcription factors involved in lipid accumulation in Yarrowia lipolytica

Overexpression screen reveals transcription factors involved in lipid accumulation in Yarrowia... Abstract Yarrowia lipolytica is a non-conventional oleaginous yeast that displays high lipid titers and yields; its production capacity holds significant promise for industrial biolipid applications. While its lipid metabolism has been widely studied, little is known about its transcriptional regulatory network. Deciphering the role of transcriptional regulators is crucial for understanding lipid accumulation, a complex phenomenon. To identify the transcription factors involved in lipid metabolism, we developed a systematic overexpression approach for 148 putative transcription factors. Analyses of overexpressing transformants revealed that 38 had an impact on lipid accumulation under at least one of the growth conditions tested. For most of these factors, our results provide the first experimentally determined functional annotation. Our data suggest that the regulation network differs depending on the carbon source, which is critical information when carrying out industrial bioprocesses. These results will therefore help guide further rational metabolic engineering for improving biolipid production by Y. lipolytica. Moreover, this work has created the largest collection of Y. lipolytica overexpressing strains to date, which will be useful in phenotype screening. lipids, overexpression, screening, transcription factors, Yarrowia lipolytica, gateway INTRODUCTION Yarrowia lipolytica is a non-conventional oleaginous yeast that is phylogenetically distant from the more conventional Saccharomyces cerevisiae (Dujon et al.2004). It is capable of accumulating large amounts of lipids (Dulermo and Nicaud 2011; Blazeck et al.2014; Qiao et al.2015, 2017), and, it is used to produce typical and atypical lipids by metabolic engineering (Ledesma-Amaro and Nicaud 2016). These advantageous features have led to the recent development of multiple modern genetic tools and technologies specific for Y. lipolytica, such as the Gateway systematic overexpression system, the Golden Gate assembly system, and a CRISPR/Cas9 gene-editing system (Leplat, Nicaud and Rossignol 2015; Gao et al.2016, Schwartz et al.2016; Celinska et al.2017; Rigouin et al.2017; Schwartz et al.2017), which have improved high-throughput screening and allowed fine-tuning of metabolic pathways. Moreover, several genome sequences from different strains are now available (Dujon et al.2004; Liu and Alper 2014; Devillers et al.2016; Magnan et al.2016). Yarrowia lipolytica has thus become a key microorganism used in both industrial applications and fundamental lipid metabolism research. However, the utility of all these tools relies on knowledge of Y. lipolytica’s metabolic pathways and gene functions, which remains incomplete as they are adapted for targeted modification and not developed yet for large-scale and systematic modifications. Many gene functions in Y. lipolytica have been characterized, thanks to the knock-out approach. However, there are still numerous genes with unknown functions, only 8% of the CDS have a proper name with a function that remains putative for a part of them (http://gryc.inra.fr/). Exploration of the regulatory network has just begun (Pomraning et al.2016; Kerkhoven et al.2017; Trébulle et al.2017) and many cellular processes remain underexplored. To fill the gaps in knowledge for this non-conventional yeast, large-scale transformant construction and phenotype determination are required. One challenge is that systematic gene deletion is tedious and time consuming in this yeast because the non-homologous end-joining pathway is favored in the repair of DNA double-strand breaks. On the other hand, tools for constructing strain collections via overexpression strategy have recently been developed for Y. lipolytica (Leplat, Nicaud and Rossignol 2015). Overexpression allows the targeting of genes that are problematic to delete, either because they have crucial functions or because they are functionally redundant. This approach has proven to be highly successful in large-scale analyses in S. cerevisiae and has led to the functional characterization of transcription factors (TFs) and signaling pathways (Stevenson, Kennedy and Harlow 2001; Chua et al.2006; Sopko et al.2006; Jin et al.2008). For example, the filamentation process in S. cerevisiae (Jin et al.2008; Shively et al.2013) and Candida albicans (Chauvel et al.2012) has now been extensively described thanks to this technique. Recently, a first attempt was made in Y. lipolytica to carry out a low-throughput overexpression of 44 genes known to be involved in lipid metabolism (Silverman et al.2016); the aim was to identify the most metabolically influential genes. In another study, a cDNA library from Y. lipolytica was overexpressed in S. cerevisiae to identify specific genes that could improve free fatty acid production in the latter species (Shi et al.2016). Overexpression is particularly helpful for exploring TFs. Genetic redundancy among TFs due to overlapping targets means deletion is problematic (Hughes and de Boer 2013). Moreover, many TFs are inactive during the stationary phase (Chua et al.2006; Jin et al.2008) and under standard lab conditions more generally (Hughes and de Boer 2013). Consequently, while deletion could easily fail to reveal phenotypes and functions, overexpression should have a greater likelihood of success. In general, TFs constitute a major category of proteins that are phosphoregulated during nitrogen starvation and lipid accumulation (Pomraning et al.2016), which means their genes are ideal targets for a large-scale overexpression screening strategy seeking to decipher the regulation of lipid accumulation in Y. lipolytica. As the transcriptional regulatory network has undergone significant evolutionary restructuring in fungi, including in the distantly related Y. lipolytica (Lavoie, Hogues and Whiteway 2009; Lavoie et al.2010; Maguire et al.2014), it would be misguided to assume that the TF regulatory network and TF functions in S. cerevisiae reflect those in other yeast. However, in Y. lipolytica, only a few TFs have been studied in the context of lipid metabolism. They include Por1, a zinc finger TF ortholog of farA in Aspergillus nidulans (Poopanitpan et al.2010); Mig1, a zinc finger TF (Wang et al.2013); the basic helix-loop-helix and Opi1-like TFs (i.e. Yas1, Yas2 and Yas3) (Endoh-Yamagami et al.2007; Hirakawa et al.2009; Kobayashi et al.2015); and, more recently, four genuine GATA-binding zinc finger (GZF) proteins and two GATA-like binding proteins (i.e. genes gzf1 to gzf6) (Pomraning, Bredeweg and Baker 2017). The putative roles of most Y. lipolytica TFs remain unknown or undescribed. There are many different classes of TFs in general and around 80 exist in fungi (Shelest 2017). It is currently estimated that the S. cerevisiae genome contains approximately 200 TF genes (Hughes and de Boer 2013). Even if S. cerevisiae is distant from Y. lipolytica, they have roughly the same number of coding sequences. We can speculate that the number of TFs in Y. lipolytica is in the same order of magnitude. Here, we developed a systematic overexpression approach in Y. lipolytica to identify putative TFs involved in lipid metabolism. We identified 148 putative TFs in the Y. lipolytica’s genome; we obtained 125 yeast transformant strains that each overexpressed a putative TF; and we found that 38 of the TFs had an impact on lipid accumulation under at least one growth condition. MATERIALS AND METHODS Identification of putative TFs All the coding sequences for Y. lipolytica were extracted from the GRYC database (http://gryc.inra.fr/). We first searched for descriptions of TFs in the annotations. We also searched for putative TF motifs using the PFAM database (http://pfam.xfam.org/); the threshold was a PFAM significance of one. To extend the list of putative TFs, known S. cerevisiae TFs were BLASTP searched against the Y. lipolytica proteome. All putative TFs previously identified in Y. lipolytica using alternative approaches were also included. The TF-specific PFAM domains that were identified in Y. lipolytica coding sequences are provided in Table S1, Supporting Information. Gateway cloning of putative TFs TFs were cloned in Gateway donor vectors and transferred to our Gateway expression vector JMP1529 (Leplat, Nicaud and Rossignol 2015) using Gateway LR clonase (Thermo Fisher Scientific, Villebon-sur-Yvette, France) in accordance with the manufacturer's instructions. JMP1529 is an integrative vector that allows, after LR recombination with donor vectors, to place genes under the control of the strong pTEF promoter. The donor vectors had one of two origins. First, some came from a sequenced cDNA library constructed with the CloneMiner cDNA Library Construction Kit (Thermo Fisher Scientific) that provides cDNA in a Gateway Entry vector (Mekouar et al.2010). Second, if they were not available from the collection, they were PCR amplified from genomic DNA and cloned using a pENTR Directional TOPO® Cloning Kit (Thermo Fisher Scientific) in accordance with the manufacturer's instructions. The origin of the TF cloning methods as well as the primer sequences for genomic DNA amplification (when applicable) are indicated in Table S1, Supporting Information. Yarrowia lipolytica transformation Yarrowia lipolytica strain JMY2566 was used in overexpression cassette transformation employing the recently developed high-throughput method for Y. lipolytica transformant library screening (Leplat, Nicaud and Rossignol 2015). When the first transformation failed, subsequent transformations were carried out using the standard lithium acetate method (Le Dall, Nicaud and Gaillardin 1994). Transformants were selected on YNB medium for URA3 complementation. Integration of the expression cassettes at the specific URA3-zeta platform in the JMY2566 genome was verified by PCR as described elsewhere (Leplat, Nicaud and Rossignol 2015). The control strain for all the experiments—JMY2810—was derived from JMY2566 by integrating an empty overexpression cassette at the URA3-zeta locus (Leplat, Nicaud and Rossignol 2015). For growth in 96 well plates, the prototroph strain JMY2900 (Dulermo et al.2014) was also used as a second control strain. Growth and media The URA + transformants were selected after incubation at 28°C on solid minimal YNB medium. YNB is composed of 0.17% (w/v) of yeast nitrogen base without ammonium and without amino acids, 0.5% (w/v) of ammonium chloride, 50 mM phosphate buffer (pH 6.8) and 1% (w/v) glucose, complemented with 1.5% (w/v) agar for solid media. Growth screening was performed using 96-well plates; 5 μL of a 24-h YPD (1% yeast extract, 1% Bacto Peptone, 2% glucose) pre-culture was used to inoculate 195 μL of YNB medium that had either 1% glucose or 1% glycerol. These compounds are the two major carbon sources used by Y. lipolytica. Cultures were grown at 28°C under constant agitation on a Biotek Synergy MX microtiter plate reader (Biotek Instruments, Colmar, France); growth was monitored by measuring optical density (OD) at 600 nm every 10 min for 24 h. Above that time in microtiter plate reader (around OD = 1.2), the correlation between OD and cell density is not linear anymore. Each treatment was performed in duplicate in separate experiments. To characterize transformant growth, maximum growth rate (Vmax, OD h−1) and maximum cell density (max OD) were calculated using a sliding eight-point scale using GEN5 2.0 software (Biotek Instruments). We considered that growth had been affected when strains displayed a mean 20% increase or decrease in Vmax or max OD relative to the median values of the six control strains present on the same plate. To determine lipid production, cultures were grown in 250 mL flasks in 25 mL YNB media containing either 3% glucose or 3% glycerol as the carbon source; the nitrogen concentration was adjusted to obtain a carbon-to-nitrogen ratio of 30 (C/N 30) for optimal lipid production (Gajdoš et al.2016). Cultures were sampled after 72 h of growth. Flask cultures were performed by batch of 20 to 30 flasks. A control strain (JMY2810) was systematically run in each batch of culture and lipids were extracted and analyzed at the same time. Samples were compared to the control strain of the same batch to avoid technical bias. Each transformant was cultured twice in separate series. Therefore, we compare percentage of variation reported to control strain used in the batch, rather than absolute data. Quantification of total lipid content Cells were harvested, washed with deionized water and freeze-dried. Fatty acids from an aliquot of lyophilized biomass (10–20 mg) were converted to their fatty acid methyl esters (FAMEs) using the Browse method (Browse, McCourt and Somerville 1986). Dried cells were mixed with 1 mL of 2.5% (v/v) sulfuric acid in methanol, which contained 50 μg of dodecanoic acid (Sigma, Saint Louis, MO, USA) as an internal standard, and were then incubated at 80°C for 90 min to form FAMEs. After transesterification, 1.5 mL of water was added, and FAMEs were extracted through the addition of 450 μL of hexane. The FAME-containing hexane phase was analyzed by gas chromatography using a Varian 3900 unit equipped with a flame ionization detector and a Varian FactorFour vf-23ms column, where the bleed specification at 260°C was 3 pA (30 m, 0.25 mm, 0.25 μm). Fatty acids were identified by comparison to commercial FAME standards (FAME32; Supelco, Sigma Aldrich, Saint-Quentin Fallavier, France) and quantified using the internal standard. RESULTS TF identification and construction of overexpression strains Few TFs have been experimentally described in Y. lipolytica. It is important to note that the definition of a TF is dependent on the exact criteria used, which leads to large discrepancies in TF identification. To compile a relatively exhaustive and highly reliable TF library before initiating cloning, several strategies were employed. First, we searched the literature and gene descriptions in genomic databases for known TFs. We then identified additional putative TFs using a PFAM domain search and a BLASTP comparison, as described in the Methods. We ended up with a list of 148 putative TFs for the Y. lipolytica genome (Table S1, Supporting Information). All 148 putative TFs were successfully cloned using our Gateway overexpression system and overexpression cassettes were used to transform the strain JMY2566. The transformation process created 125 different overexpressing strains—a cloning success rate of over 85% (i.e. from initial cloning to yeast transformation). The 23 other overexpression cassettes that failed to produce transformants were included in repeated but unsuccessful transformation procedures (5 or more repetitions). We believe that overexpression of these TFs is somehow toxic and results in transformant death. Looking for potential orthologs in S. cerevisiae does not reveal any GO enrichment neither for overepressed or knock out homologs phenotype. However, the weak similarity observed for most of these genes with S. cerevisiae orthologs do not allow to properly detect any enrichment. All transformants were tested by PCR to confirm the integration of the overexpression cassette at the URA3-zeta locus (Leplat, Nicaud and Rossignol 2015). There was a 70% rate of integration at the appropriate locus (89/125) (Table S1, Supporting Information). In the other transformants, integration occurred at random genome locations which could alter the level of expression of the overexpressed gene and may affect the detection level of possible phenotype. Overexpression has been confirmed for the mutant overexpressing TF072 (YALI0C22682g), with an increase fold change of 9 compare to control strain (Fig. S1, Supporting Information). Transformant growth As TFs can have a wide range of effects on cellular processes, we first checked our collection of TF-overexpressing strains to identify mutants with possible growth defects. To obtain precise growth kinetics, all transformants were screened on 96-well plates using glucose or glycerol in a 96-well plate reader rather than making single point measurements in flask which might only reflect maximum OD differences. Even if these growth conditions are slightly different from the flask cultures performed for lipid content measurement in term of agitation and oxygenation, it gives an overview of growth perturbation potentially generated by the gene overexpression. Based on maximum growth rate and cell density (see Methods and Table S2, Supporting Information), we identified four transformants whose growth was affected in glucose medium (i.e. decreased growth: TF072, TF079, TF099 and TF116) (Fig. 1A) and nine mutants whose growth was affected in glycerol medium (i.e. decreased growth: TF069, TF112 and TF131; increased growth: TF037, TF073, TF085, TF089, TF090 and TF091) (examples given in Fig. 1B). Most of these transformants were found to have near-threshold growth rates and cell densities on the opposing medium, indicating that for most of them, the overexpression may have a general effect on growth but is modulated by the carbon source. Figure 1. View largeDownload slide (A) Growth in glucose medium of the four mutants with glucose-growth defects (96-well plates). (B) Growth in glycerol medium of some representative mutants with glycerol-growth increase or decrease (96-well plates). Figure 1. View largeDownload slide (A) Growth in glucose medium of the four mutants with glucose-growth defects (96-well plates). (B) Growth in glycerol medium of some representative mutants with glycerol-growth increase or decrease (96-well plates). Analysis of total lipid content The ability of the TF-overexpressing strains to accumulate lipids was assessed after 72 h of growth in glucose or glycerol medium. Comparisons with the control strain allowed us to identify transformants whose lipid-accumulation phenotype was affected (Table S3, Supporting Information). In Fig. 2, we have highlighted the transformants whose lipid accumulation abilities changed the most; our threshold was a 15% increase or decrease in lipid content relative to that of the control strain (this later ranging from 8% to 10% of the dry cell weight). In glucose medium, we identified six mutants with decreased lipid content and 20 mutants with increased lipid content; the % change ranged from –26.6% (TF124; YALI0A16841g) to +90.9% (TF053; YALI0E31757g) (Fig. 2A; Table 1). In glycerol medium, we identified 13 mutants with decreased lipid content and 7 mutants with increased lipid content; the % change ranged from –55.7% (TF099; YALI0A18469g) to +74% (TF108; YALI0F25861g) (Fig. 2B; Table 1). Three transformants had increased lipid content in both media (TF019; YALI0C13178g, TF084; YALI0B08734g and TF072; YALI0C22682g), and two had decreased lipid content in both media (TF099; YALI0A18469g and TF107; YALI0C13750g). Two presented a growth defect in both glucose and glycerol media, albeit to a lesser extent in the latter case (TF072; YALI0C22682g and TF099; YALI0A18469g) (Table S2, Supporting Information). Overall, there was a weak correlation in lipid content changes in glucose versus glycerol medium for the transformants (Spearman's rank correlation: rs = 0.1539). Figure 2. View largeDownload slide Overexpressing transformants displaying notable lipid-accumulation phenotypes. (A) Percentage change in lipid content by the overexpressing strains relative to the control strain when grown in glucose medium. (B) Percentage change in lipid content by the overexpressing strains relative to the control strain when grown in glycerol medium. Figure 2. View largeDownload slide Overexpressing transformants displaying notable lipid-accumulation phenotypes. (A) Percentage change in lipid content by the overexpressing strains relative to the control strain when grown in glucose medium. (B) Percentage change in lipid content by the overexpressing strains relative to the control strain when grown in glycerol medium. Table 1. List of overexpressing transformants displaying lipid-accumulation phenotypes. Glucose Percentage change in lipid content (relative to control)a TF number ID Mean Std deviation Description Name TF124 YALI0A16841g –26.6 1.00 Conserved hypothetical protein TF099 YALI0A18469g –23.3 1.34 Homeobox protein HOY1 TF107 YALI0C13750g –20.8 1.11 Conserved hypothetical protein some similarities with S. cerevisiae YKL062W MSN4 transcriptional activator TF127 YALI0E18304g –17.5 2.01 Conserved hypothetical protein some similarities with S. cerevisiae YBR239c Putative 60.3 kDa transcriptional regulatory protein TF112 YALI0C19063g –16.9 0.68 Conserved hypothetical protein weakly similar to Nectria haematococca hypothetical protein NECHADRAFT_43519 TF051 YALI0E31383g –15.5 0.85 Conserved hypothetical protein weakly similar to Neurospora crassa hypothetical protein TF046 YALI0E17721g 16.7 0.92 Conserved hypothetical protein similar to Spathaspora passalidarum SPAPADRAFT_137815 hypothetical protein TF003 YALI0B00660g 18.8 0.64 Conserved hypothetical protein weakly similar to S. cerevisiae YDR213W UPC2 regulatory protein involved in control of sterol uptake TF059 YALI0F06072g 19.2 0.85 Conserved hypothetical protein some similarities with N. crassa Probable cutinase transcription factor 1 beta TF071 YALI0E10131g 20.8 3.54 Transcription elongation factor with a conserved zinc finger domain, similar to S. cerevisiae YKL160W ELF1 Transcription elongation factor with a conserved zinc finger domain TF072 YALI0C22682g 21.0 0.57 Conserved hypothetical protein some similarities with S. cerevisiae YJL110C GZF3 transcriptional repressor GZF3 TF074 YALI0D09757g 21.9 0.14 Conserved hypothetical protein weakly similar to S. cerevisiae YHL009c YAP3 transcription factor, of a fungal-specific family of bzip proteins TF128 YALI0F05896g 22.5 1.27 Conserved hypothetical protein weakly similar to Tuber melanosporum hypothetical protein TF040 YALI0E03410g 22.6 0.76 Conserved hypothetical protein weakly similar to S. cerevisiae YBR239c TF085 YALI0C11858g 28.0 0.92 Gal4-type transcription factor, weakly similar to Y. lipolytica YALI1E31383g, putative sequence-specific DNA binding RNA polymerase II transcription factor TF139 Yali0E05577g 28.3 1.48 Hypothetical protein of a 6-member family, conserved in the Yarrowia clade highly similar to Y. lipolytica YALI1F13761g, protein with HXXEE motif TF055 YALI0F03157g 32.6 0.92 Conserved hypothetical protein some similarities with S. cerevisiae YDR253C MET32 transcriptional regulator of sulfur amino acid metabolism TF135 Yali0E14971g 35.6 4.88 Conserved hypothetical protein some similarities with S. cerevisiae YGR044c Zinc finger protein RME1 and other transcription factors TF109 YALI0E16577g 37.2 4.31 Conserved hypothetical protein Forward some similarities with S. cerevisiae YKL185w ASH1 negative regulator of HO transcription GZF5 TF019 YALI0C13178g 41.0 3.54 Conserved hypothetical protein some similarities with S. cerevisiae YOR344c TYE7 basic helix-loop-helix transcription factor TF057 YALI0F05104g 43.2 6.72 Conserved hypothetical protein weakly similar to S. cerevisiae YPR186c TFC2 TFIIIA transcription initiation factor TF084 YALI0B08734g 50.5 3.25 Cytoplasmic pre-60S factor, putative similar to S. cerevisiae YBR267W REI1 Cytoplasmic pre-60S factor TF064 YALI0F16599g 53.5 1.13 Conserved hypothetical protein weakly similar to Debaryomyces hansenii DEHA2B03564p TF086 YALI0B04510g 57.2 2.97 Conserved hypothetical weakly similar to S. cerevisiae YKL032C IXR1 intrastrand crosslink recognition protein and transcription factor TF077 YALI0B08206g 85.7 1.98 Y. lipolytica YALI1B08206g CRF1 Copper resistance protein CRF1 TF053 YALI0E31757g 90.9 6.15 Conserved hypothetical protein weakly similar to N. crassa 123A4.250 Related to NsdD protein TF099 YALI0A18469g –55.75 24.40 Homeobox protein HOY1 TF107 YALI0C13750g –41.65 1.63 Conserved hypothetical protein some similarities with S. cerevisiae YKL062W MSN4 transcriptional activator TF089 YALI0C06842g –35.2 11.31 Transcription factor, putative similar to S. cerevisiae YMR043W MCM1 Transcription factor involved in cell-type-specific transcription and pheromone response TF073 YALI0B15818g –34.8 3.25 Conserved hypothetical protein some similarities with S. cerevisiae YDR213W UPC2 Sterol regulatory element binding protein TF050 YALI0E30789g –31.25 3.75 Conserved hypothetical protein weakly similar to Ajellomyces dermatitidis C2H2 finger domain-containing protein TF083 YALI0C02387g –31 1.56 Y. lipolytica YALI1C02387g YAS1 Basic helix-loop-helix transcription factor essential for cytochrome p450 induction in response to alkanes YAS1 TF068 YALI0E13948g –28.65 4.88 Conserved hypothetical protein some similarities with S. cerevisiae YGL073w HSF1 heat shock transcription factor TF076 YALI0D02475g –27.3 1.27 Conserved hypothetical protein weakly similar to Sclerotinia sclerotiorum hypothetical protein SS1G_03399 TF109 YALI0E16577g –23.45 3.89 Conserved hypothetical protein some similarities with S. cerevisiae YKL185w ASH1 negative regulator of HO transcription GZF5 TF098 YALI0D01463g –22.2 1.56 Conserved hypothetical protein some similarities with S. cerevisiae YNL027w CRZ1 calcineurin responsive zinc finger TF064 YALI0F16599g –20.7 3.68 Conserved hypothetical protein weakly similar to D. hansenii DEHA2B03564p TF075 YALI0F05346g –16.1 0.14 Conserved hypothetical protein weakly similar to Fusarium solani Cutinase gene palindrome-binding protein TF077 YALI0B08206g –16.05 0.07 Y. lipolytica YALI1B08206g CRF1 Copper resistance protein CRF1 TF039 YALI0D23749g 17.95 1.63 Conserved hypothetical protein weakly similar to S. cerevisiae YJL056c ZAP1 metalloregulatory protein involved in zinc-responsive transcriptional regulation TF079 YALI0D12628g 18.05 0.07 Y. lipolytica YALI1D12628g POR1 Subunit of the SWI/SNF chromatin remodeling complex involved in transcriptional regulation POR1 TF019 YALI0C13178g 18.3 0.57 Conserved hypothetical protein some similarities with S. cerevisiae YOR344c TYE7 basic helix-loop-helix transcription factor TF084 YALI0B08734g 18.45 0.07 Cytoplasmic pre-60S factor, similar to S. cerevisiae YBR267W REI1 Cytoplasmic pre-60S factor TF072 YALI0C22682g 22.4 4.53 Conserved hypothetical protein some similarities with S. cerevisiae YJL110C GZF3 transcriptional repressor GZF3 TF001 YALI0A10637g 27.3 1.13 Conserved hypothetical protein TF108 YALI0F25861g 74 22.34 Conserved hypothetical protein weakly similar to S. cerevisiae YDL020c SON1 26S proteasome subunit Glucose Percentage change in lipid content (relative to control)a TF number ID Mean Std deviation Description Name TF124 YALI0A16841g –26.6 1.00 Conserved hypothetical protein TF099 YALI0A18469g –23.3 1.34 Homeobox protein HOY1 TF107 YALI0C13750g –20.8 1.11 Conserved hypothetical protein some similarities with S. cerevisiae YKL062W MSN4 transcriptional activator TF127 YALI0E18304g –17.5 2.01 Conserved hypothetical protein some similarities with S. cerevisiae YBR239c Putative 60.3 kDa transcriptional regulatory protein TF112 YALI0C19063g –16.9 0.68 Conserved hypothetical protein weakly similar to Nectria haematococca hypothetical protein NECHADRAFT_43519 TF051 YALI0E31383g –15.5 0.85 Conserved hypothetical protein weakly similar to Neurospora crassa hypothetical protein TF046 YALI0E17721g 16.7 0.92 Conserved hypothetical protein similar to Spathaspora passalidarum SPAPADRAFT_137815 hypothetical protein TF003 YALI0B00660g 18.8 0.64 Conserved hypothetical protein weakly similar to S. cerevisiae YDR213W UPC2 regulatory protein involved in control of sterol uptake TF059 YALI0F06072g 19.2 0.85 Conserved hypothetical protein some similarities with N. crassa Probable cutinase transcription factor 1 beta TF071 YALI0E10131g 20.8 3.54 Transcription elongation factor with a conserved zinc finger domain, similar to S. cerevisiae YKL160W ELF1 Transcription elongation factor with a conserved zinc finger domain TF072 YALI0C22682g 21.0 0.57 Conserved hypothetical protein some similarities with S. cerevisiae YJL110C GZF3 transcriptional repressor GZF3 TF074 YALI0D09757g 21.9 0.14 Conserved hypothetical protein weakly similar to S. cerevisiae YHL009c YAP3 transcription factor, of a fungal-specific family of bzip proteins TF128 YALI0F05896g 22.5 1.27 Conserved hypothetical protein weakly similar to Tuber melanosporum hypothetical protein TF040 YALI0E03410g 22.6 0.76 Conserved hypothetical protein weakly similar to S. cerevisiae YBR239c TF085 YALI0C11858g 28.0 0.92 Gal4-type transcription factor, weakly similar to Y. lipolytica YALI1E31383g, putative sequence-specific DNA binding RNA polymerase II transcription factor TF139 Yali0E05577g 28.3 1.48 Hypothetical protein of a 6-member family, conserved in the Yarrowia clade highly similar to Y. lipolytica YALI1F13761g, protein with HXXEE motif TF055 YALI0F03157g 32.6 0.92 Conserved hypothetical protein some similarities with S. cerevisiae YDR253C MET32 transcriptional regulator of sulfur amino acid metabolism TF135 Yali0E14971g 35.6 4.88 Conserved hypothetical protein some similarities with S. cerevisiae YGR044c Zinc finger protein RME1 and other transcription factors TF109 YALI0E16577g 37.2 4.31 Conserved hypothetical protein Forward some similarities with S. cerevisiae YKL185w ASH1 negative regulator of HO transcription GZF5 TF019 YALI0C13178g 41.0 3.54 Conserved hypothetical protein some similarities with S. cerevisiae YOR344c TYE7 basic helix-loop-helix transcription factor TF057 YALI0F05104g 43.2 6.72 Conserved hypothetical protein weakly similar to S. cerevisiae YPR186c TFC2 TFIIIA transcription initiation factor TF084 YALI0B08734g 50.5 3.25 Cytoplasmic pre-60S factor, putative similar to S. cerevisiae YBR267W REI1 Cytoplasmic pre-60S factor TF064 YALI0F16599g 53.5 1.13 Conserved hypothetical protein weakly similar to Debaryomyces hansenii DEHA2B03564p TF086 YALI0B04510g 57.2 2.97 Conserved hypothetical weakly similar to S. cerevisiae YKL032C IXR1 intrastrand crosslink recognition protein and transcription factor TF077 YALI0B08206g 85.7 1.98 Y. lipolytica YALI1B08206g CRF1 Copper resistance protein CRF1 TF053 YALI0E31757g 90.9 6.15 Conserved hypothetical protein weakly similar to N. crassa 123A4.250 Related to NsdD protein TF099 YALI0A18469g –55.75 24.40 Homeobox protein HOY1 TF107 YALI0C13750g –41.65 1.63 Conserved hypothetical protein some similarities with S. cerevisiae YKL062W MSN4 transcriptional activator TF089 YALI0C06842g –35.2 11.31 Transcription factor, putative similar to S. cerevisiae YMR043W MCM1 Transcription factor involved in cell-type-specific transcription and pheromone response TF073 YALI0B15818g –34.8 3.25 Conserved hypothetical protein some similarities with S. cerevisiae YDR213W UPC2 Sterol regulatory element binding protein TF050 YALI0E30789g –31.25 3.75 Conserved hypothetical protein weakly similar to Ajellomyces dermatitidis C2H2 finger domain-containing protein TF083 YALI0C02387g –31 1.56 Y. lipolytica YALI1C02387g YAS1 Basic helix-loop-helix transcription factor essential for cytochrome p450 induction in response to alkanes YAS1 TF068 YALI0E13948g –28.65 4.88 Conserved hypothetical protein some similarities with S. cerevisiae YGL073w HSF1 heat shock transcription factor TF076 YALI0D02475g –27.3 1.27 Conserved hypothetical protein weakly similar to Sclerotinia sclerotiorum hypothetical protein SS1G_03399 TF109 YALI0E16577g –23.45 3.89 Conserved hypothetical protein some similarities with S. cerevisiae YKL185w ASH1 negative regulator of HO transcription GZF5 TF098 YALI0D01463g –22.2 1.56 Conserved hypothetical protein some similarities with S. cerevisiae YNL027w CRZ1 calcineurin responsive zinc finger TF064 YALI0F16599g –20.7 3.68 Conserved hypothetical protein weakly similar to D. hansenii DEHA2B03564p TF075 YALI0F05346g –16.1 0.14 Conserved hypothetical protein weakly similar to Fusarium solani Cutinase gene palindrome-binding protein TF077 YALI0B08206g –16.05 0.07 Y. lipolytica YALI1B08206g CRF1 Copper resistance protein CRF1 TF039 YALI0D23749g 17.95 1.63 Conserved hypothetical protein weakly similar to S. cerevisiae YJL056c ZAP1 metalloregulatory protein involved in zinc-responsive transcriptional regulation TF079 YALI0D12628g 18.05 0.07 Y. lipolytica YALI1D12628g POR1 Subunit of the SWI/SNF chromatin remodeling complex involved in transcriptional regulation POR1 TF019 YALI0C13178g 18.3 0.57 Conserved hypothetical protein some similarities with S. cerevisiae YOR344c TYE7 basic helix-loop-helix transcription factor TF084 YALI0B08734g 18.45 0.07 Cytoplasmic pre-60S factor, similar to S. cerevisiae YBR267W REI1 Cytoplasmic pre-60S factor TF072 YALI0C22682g 22.4 4.53 Conserved hypothetical protein some similarities with S. cerevisiae YJL110C GZF3 transcriptional repressor GZF3 TF001 YALI0A10637g 27.3 1.13 Conserved hypothetical protein TF108 YALI0F25861g 74 22.34 Conserved hypothetical protein weakly similar to S. cerevisiae YDL020c SON1 26S proteasome subunit aThe strains that showed notably decreased and increased lipid contents are in green and red, respectively. View Large Table 1. List of overexpressing transformants displaying lipid-accumulation phenotypes. Glucose Percentage change in lipid content (relative to control)a TF number ID Mean Std deviation Description Name TF124 YALI0A16841g –26.6 1.00 Conserved hypothetical protein TF099 YALI0A18469g –23.3 1.34 Homeobox protein HOY1 TF107 YALI0C13750g –20.8 1.11 Conserved hypothetical protein some similarities with S. cerevisiae YKL062W MSN4 transcriptional activator TF127 YALI0E18304g –17.5 2.01 Conserved hypothetical protein some similarities with S. cerevisiae YBR239c Putative 60.3 kDa transcriptional regulatory protein TF112 YALI0C19063g –16.9 0.68 Conserved hypothetical protein weakly similar to Nectria haematococca hypothetical protein NECHADRAFT_43519 TF051 YALI0E31383g –15.5 0.85 Conserved hypothetical protein weakly similar to Neurospora crassa hypothetical protein TF046 YALI0E17721g 16.7 0.92 Conserved hypothetical protein similar to Spathaspora passalidarum SPAPADRAFT_137815 hypothetical protein TF003 YALI0B00660g 18.8 0.64 Conserved hypothetical protein weakly similar to S. cerevisiae YDR213W UPC2 regulatory protein involved in control of sterol uptake TF059 YALI0F06072g 19.2 0.85 Conserved hypothetical protein some similarities with N. crassa Probable cutinase transcription factor 1 beta TF071 YALI0E10131g 20.8 3.54 Transcription elongation factor with a conserved zinc finger domain, similar to S. cerevisiae YKL160W ELF1 Transcription elongation factor with a conserved zinc finger domain TF072 YALI0C22682g 21.0 0.57 Conserved hypothetical protein some similarities with S. cerevisiae YJL110C GZF3 transcriptional repressor GZF3 TF074 YALI0D09757g 21.9 0.14 Conserved hypothetical protein weakly similar to S. cerevisiae YHL009c YAP3 transcription factor, of a fungal-specific family of bzip proteins TF128 YALI0F05896g 22.5 1.27 Conserved hypothetical protein weakly similar to Tuber melanosporum hypothetical protein TF040 YALI0E03410g 22.6 0.76 Conserved hypothetical protein weakly similar to S. cerevisiae YBR239c TF085 YALI0C11858g 28.0 0.92 Gal4-type transcription factor, weakly similar to Y. lipolytica YALI1E31383g, putative sequence-specific DNA binding RNA polymerase II transcription factor TF139 Yali0E05577g 28.3 1.48 Hypothetical protein of a 6-member family, conserved in the Yarrowia clade highly similar to Y. lipolytica YALI1F13761g, protein with HXXEE motif TF055 YALI0F03157g 32.6 0.92 Conserved hypothetical protein some similarities with S. cerevisiae YDR253C MET32 transcriptional regulator of sulfur amino acid metabolism TF135 Yali0E14971g 35.6 4.88 Conserved hypothetical protein some similarities with S. cerevisiae YGR044c Zinc finger protein RME1 and other transcription factors TF109 YALI0E16577g 37.2 4.31 Conserved hypothetical protein Forward some similarities with S. cerevisiae YKL185w ASH1 negative regulator of HO transcription GZF5 TF019 YALI0C13178g 41.0 3.54 Conserved hypothetical protein some similarities with S. cerevisiae YOR344c TYE7 basic helix-loop-helix transcription factor TF057 YALI0F05104g 43.2 6.72 Conserved hypothetical protein weakly similar to S. cerevisiae YPR186c TFC2 TFIIIA transcription initiation factor TF084 YALI0B08734g 50.5 3.25 Cytoplasmic pre-60S factor, putative similar to S. cerevisiae YBR267W REI1 Cytoplasmic pre-60S factor TF064 YALI0F16599g 53.5 1.13 Conserved hypothetical protein weakly similar to Debaryomyces hansenii DEHA2B03564p TF086 YALI0B04510g 57.2 2.97 Conserved hypothetical weakly similar to S. cerevisiae YKL032C IXR1 intrastrand crosslink recognition protein and transcription factor TF077 YALI0B08206g 85.7 1.98 Y. lipolytica YALI1B08206g CRF1 Copper resistance protein CRF1 TF053 YALI0E31757g 90.9 6.15 Conserved hypothetical protein weakly similar to N. crassa 123A4.250 Related to NsdD protein TF099 YALI0A18469g –55.75 24.40 Homeobox protein HOY1 TF107 YALI0C13750g –41.65 1.63 Conserved hypothetical protein some similarities with S. cerevisiae YKL062W MSN4 transcriptional activator TF089 YALI0C06842g –35.2 11.31 Transcription factor, putative similar to S. cerevisiae YMR043W MCM1 Transcription factor involved in cell-type-specific transcription and pheromone response TF073 YALI0B15818g –34.8 3.25 Conserved hypothetical protein some similarities with S. cerevisiae YDR213W UPC2 Sterol regulatory element binding protein TF050 YALI0E30789g –31.25 3.75 Conserved hypothetical protein weakly similar to Ajellomyces dermatitidis C2H2 finger domain-containing protein TF083 YALI0C02387g –31 1.56 Y. lipolytica YALI1C02387g YAS1 Basic helix-loop-helix transcription factor essential for cytochrome p450 induction in response to alkanes YAS1 TF068 YALI0E13948g –28.65 4.88 Conserved hypothetical protein some similarities with S. cerevisiae YGL073w HSF1 heat shock transcription factor TF076 YALI0D02475g –27.3 1.27 Conserved hypothetical protein weakly similar to Sclerotinia sclerotiorum hypothetical protein SS1G_03399 TF109 YALI0E16577g –23.45 3.89 Conserved hypothetical protein some similarities with S. cerevisiae YKL185w ASH1 negative regulator of HO transcription GZF5 TF098 YALI0D01463g –22.2 1.56 Conserved hypothetical protein some similarities with S. cerevisiae YNL027w CRZ1 calcineurin responsive zinc finger TF064 YALI0F16599g –20.7 3.68 Conserved hypothetical protein weakly similar to D. hansenii DEHA2B03564p TF075 YALI0F05346g –16.1 0.14 Conserved hypothetical protein weakly similar to Fusarium solani Cutinase gene palindrome-binding protein TF077 YALI0B08206g –16.05 0.07 Y. lipolytica YALI1B08206g CRF1 Copper resistance protein CRF1 TF039 YALI0D23749g 17.95 1.63 Conserved hypothetical protein weakly similar to S. cerevisiae YJL056c ZAP1 metalloregulatory protein involved in zinc-responsive transcriptional regulation TF079 YALI0D12628g 18.05 0.07 Y. lipolytica YALI1D12628g POR1 Subunit of the SWI/SNF chromatin remodeling complex involved in transcriptional regulation POR1 TF019 YALI0C13178g 18.3 0.57 Conserved hypothetical protein some similarities with S. cerevisiae YOR344c TYE7 basic helix-loop-helix transcription factor TF084 YALI0B08734g 18.45 0.07 Cytoplasmic pre-60S factor, similar to S. cerevisiae YBR267W REI1 Cytoplasmic pre-60S factor TF072 YALI0C22682g 22.4 4.53 Conserved hypothetical protein some similarities with S. cerevisiae YJL110C GZF3 transcriptional repressor GZF3 TF001 YALI0A10637g 27.3 1.13 Conserved hypothetical protein TF108 YALI0F25861g 74 22.34 Conserved hypothetical protein weakly similar to S. cerevisiae YDL020c SON1 26S proteasome subunit Glucose Percentage change in lipid content (relative to control)a TF number ID Mean Std deviation Description Name TF124 YALI0A16841g –26.6 1.00 Conserved hypothetical protein TF099 YALI0A18469g –23.3 1.34 Homeobox protein HOY1 TF107 YALI0C13750g –20.8 1.11 Conserved hypothetical protein some similarities with S. cerevisiae YKL062W MSN4 transcriptional activator TF127 YALI0E18304g –17.5 2.01 Conserved hypothetical protein some similarities with S. cerevisiae YBR239c Putative 60.3 kDa transcriptional regulatory protein TF112 YALI0C19063g –16.9 0.68 Conserved hypothetical protein weakly similar to Nectria haematococca hypothetical protein NECHADRAFT_43519 TF051 YALI0E31383g –15.5 0.85 Conserved hypothetical protein weakly similar to Neurospora crassa hypothetical protein TF046 YALI0E17721g 16.7 0.92 Conserved hypothetical protein similar to Spathaspora passalidarum SPAPADRAFT_137815 hypothetical protein TF003 YALI0B00660g 18.8 0.64 Conserved hypothetical protein weakly similar to S. cerevisiae YDR213W UPC2 regulatory protein involved in control of sterol uptake TF059 YALI0F06072g 19.2 0.85 Conserved hypothetical protein some similarities with N. crassa Probable cutinase transcription factor 1 beta TF071 YALI0E10131g 20.8 3.54 Transcription elongation factor with a conserved zinc finger domain, similar to S. cerevisiae YKL160W ELF1 Transcription elongation factor with a conserved zinc finger domain TF072 YALI0C22682g 21.0 0.57 Conserved hypothetical protein some similarities with S. cerevisiae YJL110C GZF3 transcriptional repressor GZF3 TF074 YALI0D09757g 21.9 0.14 Conserved hypothetical protein weakly similar to S. cerevisiae YHL009c YAP3 transcription factor, of a fungal-specific family of bzip proteins TF128 YALI0F05896g 22.5 1.27 Conserved hypothetical protein weakly similar to Tuber melanosporum hypothetical protein TF040 YALI0E03410g 22.6 0.76 Conserved hypothetical protein weakly similar to S. cerevisiae YBR239c TF085 YALI0C11858g 28.0 0.92 Gal4-type transcription factor, weakly similar to Y. lipolytica YALI1E31383g, putative sequence-specific DNA binding RNA polymerase II transcription factor TF139 Yali0E05577g 28.3 1.48 Hypothetical protein of a 6-member family, conserved in the Yarrowia clade highly similar to Y. lipolytica YALI1F13761g, protein with HXXEE motif TF055 YALI0F03157g 32.6 0.92 Conserved hypothetical protein some similarities with S. cerevisiae YDR253C MET32 transcriptional regulator of sulfur amino acid metabolism TF135 Yali0E14971g 35.6 4.88 Conserved hypothetical protein some similarities with S. cerevisiae YGR044c Zinc finger protein RME1 and other transcription factors TF109 YALI0E16577g 37.2 4.31 Conserved hypothetical protein Forward some similarities with S. cerevisiae YKL185w ASH1 negative regulator of HO transcription GZF5 TF019 YALI0C13178g 41.0 3.54 Conserved hypothetical protein some similarities with S. cerevisiae YOR344c TYE7 basic helix-loop-helix transcription factor TF057 YALI0F05104g 43.2 6.72 Conserved hypothetical protein weakly similar to S. cerevisiae YPR186c TFC2 TFIIIA transcription initiation factor TF084 YALI0B08734g 50.5 3.25 Cytoplasmic pre-60S factor, putative similar to S. cerevisiae YBR267W REI1 Cytoplasmic pre-60S factor TF064 YALI0F16599g 53.5 1.13 Conserved hypothetical protein weakly similar to Debaryomyces hansenii DEHA2B03564p TF086 YALI0B04510g 57.2 2.97 Conserved hypothetical weakly similar to S. cerevisiae YKL032C IXR1 intrastrand crosslink recognition protein and transcription factor TF077 YALI0B08206g 85.7 1.98 Y. lipolytica YALI1B08206g CRF1 Copper resistance protein CRF1 TF053 YALI0E31757g 90.9 6.15 Conserved hypothetical protein weakly similar to N. crassa 123A4.250 Related to NsdD protein TF099 YALI0A18469g –55.75 24.40 Homeobox protein HOY1 TF107 YALI0C13750g –41.65 1.63 Conserved hypothetical protein some similarities with S. cerevisiae YKL062W MSN4 transcriptional activator TF089 YALI0C06842g –35.2 11.31 Transcription factor, putative similar to S. cerevisiae YMR043W MCM1 Transcription factor involved in cell-type-specific transcription and pheromone response TF073 YALI0B15818g –34.8 3.25 Conserved hypothetical protein some similarities with S. cerevisiae YDR213W UPC2 Sterol regulatory element binding protein TF050 YALI0E30789g –31.25 3.75 Conserved hypothetical protein weakly similar to Ajellomyces dermatitidis C2H2 finger domain-containing protein TF083 YALI0C02387g –31 1.56 Y. lipolytica YALI1C02387g YAS1 Basic helix-loop-helix transcription factor essential for cytochrome p450 induction in response to alkanes YAS1 TF068 YALI0E13948g –28.65 4.88 Conserved hypothetical protein some similarities with S. cerevisiae YGL073w HSF1 heat shock transcription factor TF076 YALI0D02475g –27.3 1.27 Conserved hypothetical protein weakly similar to Sclerotinia sclerotiorum hypothetical protein SS1G_03399 TF109 YALI0E16577g –23.45 3.89 Conserved hypothetical protein some similarities with S. cerevisiae YKL185w ASH1 negative regulator of HO transcription GZF5 TF098 YALI0D01463g –22.2 1.56 Conserved hypothetical protein some similarities with S. cerevisiae YNL027w CRZ1 calcineurin responsive zinc finger TF064 YALI0F16599g –20.7 3.68 Conserved hypothetical protein weakly similar to D. hansenii DEHA2B03564p TF075 YALI0F05346g –16.1 0.14 Conserved hypothetical protein weakly similar to Fusarium solani Cutinase gene palindrome-binding protein TF077 YALI0B08206g –16.05 0.07 Y. lipolytica YALI1B08206g CRF1 Copper resistance protein CRF1 TF039 YALI0D23749g 17.95 1.63 Conserved hypothetical protein weakly similar to S. cerevisiae YJL056c ZAP1 metalloregulatory protein involved in zinc-responsive transcriptional regulation TF079 YALI0D12628g 18.05 0.07 Y. lipolytica YALI1D12628g POR1 Subunit of the SWI/SNF chromatin remodeling complex involved in transcriptional regulation POR1 TF019 YALI0C13178g 18.3 0.57 Conserved hypothetical protein some similarities with S. cerevisiae YOR344c TYE7 basic helix-loop-helix transcription factor TF084 YALI0B08734g 18.45 0.07 Cytoplasmic pre-60S factor, similar to S. cerevisiae YBR267W REI1 Cytoplasmic pre-60S factor TF072 YALI0C22682g 22.4 4.53 Conserved hypothetical protein some similarities with S. cerevisiae YJL110C GZF3 transcriptional repressor GZF3 TF001 YALI0A10637g 27.3 1.13 Conserved hypothetical protein TF108 YALI0F25861g 74 22.34 Conserved hypothetical protein weakly similar to S. cerevisiae YDL020c SON1 26S proteasome subunit aThe strains that showed notably decreased and increased lipid contents are in green and red, respectively. View Large DISCUSSION In this work, we setup a relatively high-throughput overexpression strategy in the yeast Y. lipolytica in order to screen for potential phenotype. While we decided to screen all proteins with a potential TF domain as they are ideal targets for a large-scale overexpression screening, our goal was not to provide evidence for TF activities but rather identification of new genes involved directly or indirectly in lipid accumulation. Nevertheless, several TFs that we targeted here have been previously studied. Historically, the first TFs studied in Y. lipolytica were related to filamentation. TF099 (YALI0A18469g) has been described as a Hoy1 homeobox protein and is required for hyphal formation in Y. lipolytica (Torres-Guzman and Dominguez 1997): a deletion of hoy1 suppresses the filamentation phenotype. When we overexpressed Hoy1, we observed a growth defect under different conditions. Microscopic observations revealed that Hoy1 overexpression induced a strong and constitutive filamentation phenotype in various media and resulted in strains with greatly reduced viability (Fig. S2, Supporting Information). This finding supports the validity of our high-throughput overexpression screening approach. Moreover, TF099 (Hoy1) was the transformant with the most pronounced decrease in lipid content in both media, which is likely linked to the filamentation phenotype. Filamentation potentially drives the remobilization of triacylglycerol in lipid droplets (Gajdoš et al.2016). To a lesser extent, MHY1 overexpression (as seen in TF095; YALI0B21582g) also resulted in a hyperfilamentous phenotype (Fig. S2, Supporting Information); Mhy1 is a C2H2-type zinc finger protein required for dimorphic transition (Hurtado and Rachubinski 1999). Only a small decrease in lipid content resulted from MHY1 overexpression (Table S3, Supporting Information). In the literature, several TFs have been reported to be involved in lipid accumulation regulation. An ortholog of farA in A. nidulans with a zinc finger domain and a fungus-specific TF domain, Por1 appears to be a transcriptional activator that regulates fatty acid metabolism in Y. lipolytica (Poopanitpan et al.2010). In the same study, three other TFs putatively involved in lipid utilization were disrupted—YALI0F13321g, YALI0D17988g and YALI0D10681g—without obtaining notable phenotypes. Our study provided confirmation of these prior results. POR1 overexpression (TF079; YALI0D12628g) supported this TF’s role in lipid accumulation, at least when yeast were grown in glycerol medium (Table 1), and the transformants overexpressing YALI0F13321g, YALI0D17988g and YALI0D10681g did not display any lipid-accumulation phenotypes (Table S3, Supporting Information). Deletion of MIG1, a zinc finger TF, has been shown to induce lipid accumulation in Y. lipolytica (Wang et al.2013). In the corresponding transformant, genes involved in lipid metabolism were derepressed, which fits with the lipid-accumulation phenotype. However, genes involved in lipid degradation were also expressed at higher levels compared to the wild type (Wang et al.2013), indicating that regulation is complex and likely operates during translation or post-translation. Accordingly, the overexpression of MIG1 (TF042; YALI0E07942g) did not affect lipid content in our screen (Table S3, Supporting Information). Yas1 and Yas2 are two basic helix-loop-helix TFs that form a heterodimer that induces ALK1, a gene involved in alkane oxidation (Endoh-Yamagami et al.2007), while Yas3, another TF, has a repressor effect (Hirakawa et al.2009). We obtained a Yas1 overexpressing mutant (TF083; YALI0C02387g) and a mutant with a spliced form of Yas3 (TF130; YALI0C14784g). Overexpression of Yas1 triggered a decrease in lipid content (Table 1), while overexpression of Yas3 did not significantly affect lipid content, suggesting they play different roles in lipid accumulation just as they do in alkane metabolism. Recently, all putative Gzf TFs were identified in Y. lipolytica. Deletion transformants were functionally characterized to determine the TFs’ roles in nitrogen catabolic repression and lipid metabolism (Pomraning, Bredeweg and Baker 2017). We also identified these six putative Gzf TFs: TF037 (Gzf1; Yali0D20482g), TF082 (Gzf2; Yali0F17886g), TF072 (Gzf3; Yali0C22682g), TF041 (Gzf4; Yali0E05555g), TF109 (Gzf5; Yali0E16577g) and TF075 (Gzf6; Yali0E05346g). We obtained overexpression transformants for five of them (TF037, TF072, TF041, TF109 and TF075). In a previous study, Δgzf2 and Δgzf3 generated higher concentrations of lipids when ammonium sulfate was the sole nitrogen source, which was not the case when peptone plus yeast extract was used (Pomraning, Bredeweg and Baker 2017). While we did not obtain a GZF2 overexpression transformant, we were able to create a GZF3-overexpressing strain (TF072), which displayed increased lipid production in both glucose and glycerol media (Fig. 2A and B; Table 1) and also had a growth defect (Fig. 1). This increase in lipid content was somewhat surprising, although we did use a different nitrogen source (i.e. ammonium chloride), which might have affected regulation via Gzf3 since regulation is nitrogen source dependent (Pomraning, Bredeweg and Baker 2017). While the deletion of GZF5 seemed to have no effect on lipid accumulation (Pomraning, Bredeweg and Baker 2017), we found here that the GZF5-overexpression mutant had increased lipid content when grown in glucose medium; the opposite was observed in glycerol medium (Fig. 2A and B; Table 1). It is one of the three transformants that displayed opposite phenotypes depending on the carbon source (Fig. 2A and B; Table 1). These results reveal that the regulation of lipid metabolism by Gzf TFs is complex. Recently, Mga2 was found to be involved in lipid accumulation (Liu et al.2015). Mga2 is not annotated as a TF and does not present a classical TF motif (but rather an immunoglobulin-like fold). It was therefore not included in the list used to create our overexpressing strain collection. Three of the indentified TFs examined here with an effect on lipid accumulation (Hoy1, Gzf3, TF057) have been described among the 10 most influential TFs during lipid accumulation by inference of coregulatory network (Trébulle et al.2017) supporting their role in this phenotype and consolidate the relevance of the results obtained in this high throughput screening. Overall, we identified 38 overexpressing strains that had altered lipid-accumulation phenotypes under at least one condition. Surprisingly, we did not observe any significant variation in term of fatty acid profiles compare to the control. Most of the putative TFs involved had not been described in the literature as affecting lipid metabolism regulation and thus their functions had not been experimentally annotated. The paucity of biological investigation for these genes makes the determination of their exact role and function very speculative at this stage. By using two different growth media in our screen, we discovered that the regulation network probably differs depending on the carbon source because a large number of the overexpressing mutants had different phenotypes in glucose versus glycerol medium. It is well established that condition-dependent changes in the binding landscape of TFs occur in S. cerevisiae, phenomena that have been highlighted by large-scale chIP-chip analyses (see Hughes and de Boer 2013 for a review). This fact indicates that complex regulatory networks can be significantly rewired based on culturing conditions. The situation in Y. lipolytica is likely similar, as would suggest the weak correlation in phenotypes observed in the two growth media. Such information is essential to have when carrying out industrial bioprocesses and should be incorporated into synthetic biology approaches in Y. lipolytica, particularly those involving biolipid production. Therefore, we need a more detailed understanding of network rewiring and genome-scale analyses should be accelerated. To our knowledge, our study is the first systematic high-throughput functional analysis of a category of genes in Y. lipolytica. We have provided information on the putative roles of an important subset of genes with up to now unknown functions. We have also built the largest collection of Y. lipolytica overexpressing strains to date, which could also be screened for other phenotypes. SUPPLEMENTARY DATA Supplementary data are available at FEMSYR online. Acknowledgements We would like to thank Heber Gamboa-Meléndez for providing the qRT-PCR data. FUNDING This work was supported by the SAS PIVERT, within the frame of the French Institute for Energy Transition (Institut pour la Transition Energétique (ITE) P.I.V.E.R.T. (www.institut-pivert.com) selected as an Investment for the Future (‘Investissements d’Avenir’)). This work was supported, as part of the Investments for the Future, by the French Government under the reference ANR-001-01. Conflict of interest. None declared. REFERENCES Blazeck J , Hill A , Liu L et al. Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production . Nat Commun 2014 ; 5 : 3131 . Google Scholar CrossRef Search ADS PubMed Browse J , McCourt PJ , Somerville CR . Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue . Anal Biochem 1986 ; 152 : 141 – 5 . Google Scholar CrossRef Search ADS PubMed Celinska E , Ledesma-Amaro R , Larroude M et al. Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica . Microb Biotechnol 2017 ; 10 : 450 – 5 . Google Scholar CrossRef Search ADS PubMed Chauvel M , Nesseir A , Cabral V et al. 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Overexpression screen reveals transcription factors involved in lipid accumulation in Yarrowia lipolytica

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Blackwell
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© FEMS 2018.
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1567-1356
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1567-1364
D.O.I.
10.1093/femsyr/foy037
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Abstract

Abstract Yarrowia lipolytica is a non-conventional oleaginous yeast that displays high lipid titers and yields; its production capacity holds significant promise for industrial biolipid applications. While its lipid metabolism has been widely studied, little is known about its transcriptional regulatory network. Deciphering the role of transcriptional regulators is crucial for understanding lipid accumulation, a complex phenomenon. To identify the transcription factors involved in lipid metabolism, we developed a systematic overexpression approach for 148 putative transcription factors. Analyses of overexpressing transformants revealed that 38 had an impact on lipid accumulation under at least one of the growth conditions tested. For most of these factors, our results provide the first experimentally determined functional annotation. Our data suggest that the regulation network differs depending on the carbon source, which is critical information when carrying out industrial bioprocesses. These results will therefore help guide further rational metabolic engineering for improving biolipid production by Y. lipolytica. Moreover, this work has created the largest collection of Y. lipolytica overexpressing strains to date, which will be useful in phenotype screening. lipids, overexpression, screening, transcription factors, Yarrowia lipolytica, gateway INTRODUCTION Yarrowia lipolytica is a non-conventional oleaginous yeast that is phylogenetically distant from the more conventional Saccharomyces cerevisiae (Dujon et al.2004). It is capable of accumulating large amounts of lipids (Dulermo and Nicaud 2011; Blazeck et al.2014; Qiao et al.2015, 2017), and, it is used to produce typical and atypical lipids by metabolic engineering (Ledesma-Amaro and Nicaud 2016). These advantageous features have led to the recent development of multiple modern genetic tools and technologies specific for Y. lipolytica, such as the Gateway systematic overexpression system, the Golden Gate assembly system, and a CRISPR/Cas9 gene-editing system (Leplat, Nicaud and Rossignol 2015; Gao et al.2016, Schwartz et al.2016; Celinska et al.2017; Rigouin et al.2017; Schwartz et al.2017), which have improved high-throughput screening and allowed fine-tuning of metabolic pathways. Moreover, several genome sequences from different strains are now available (Dujon et al.2004; Liu and Alper 2014; Devillers et al.2016; Magnan et al.2016). Yarrowia lipolytica has thus become a key microorganism used in both industrial applications and fundamental lipid metabolism research. However, the utility of all these tools relies on knowledge of Y. lipolytica’s metabolic pathways and gene functions, which remains incomplete as they are adapted for targeted modification and not developed yet for large-scale and systematic modifications. Many gene functions in Y. lipolytica have been characterized, thanks to the knock-out approach. However, there are still numerous genes with unknown functions, only 8% of the CDS have a proper name with a function that remains putative for a part of them (http://gryc.inra.fr/). Exploration of the regulatory network has just begun (Pomraning et al.2016; Kerkhoven et al.2017; Trébulle et al.2017) and many cellular processes remain underexplored. To fill the gaps in knowledge for this non-conventional yeast, large-scale transformant construction and phenotype determination are required. One challenge is that systematic gene deletion is tedious and time consuming in this yeast because the non-homologous end-joining pathway is favored in the repair of DNA double-strand breaks. On the other hand, tools for constructing strain collections via overexpression strategy have recently been developed for Y. lipolytica (Leplat, Nicaud and Rossignol 2015). Overexpression allows the targeting of genes that are problematic to delete, either because they have crucial functions or because they are functionally redundant. This approach has proven to be highly successful in large-scale analyses in S. cerevisiae and has led to the functional characterization of transcription factors (TFs) and signaling pathways (Stevenson, Kennedy and Harlow 2001; Chua et al.2006; Sopko et al.2006; Jin et al.2008). For example, the filamentation process in S. cerevisiae (Jin et al.2008; Shively et al.2013) and Candida albicans (Chauvel et al.2012) has now been extensively described thanks to this technique. Recently, a first attempt was made in Y. lipolytica to carry out a low-throughput overexpression of 44 genes known to be involved in lipid metabolism (Silverman et al.2016); the aim was to identify the most metabolically influential genes. In another study, a cDNA library from Y. lipolytica was overexpressed in S. cerevisiae to identify specific genes that could improve free fatty acid production in the latter species (Shi et al.2016). Overexpression is particularly helpful for exploring TFs. Genetic redundancy among TFs due to overlapping targets means deletion is problematic (Hughes and de Boer 2013). Moreover, many TFs are inactive during the stationary phase (Chua et al.2006; Jin et al.2008) and under standard lab conditions more generally (Hughes and de Boer 2013). Consequently, while deletion could easily fail to reveal phenotypes and functions, overexpression should have a greater likelihood of success. In general, TFs constitute a major category of proteins that are phosphoregulated during nitrogen starvation and lipid accumulation (Pomraning et al.2016), which means their genes are ideal targets for a large-scale overexpression screening strategy seeking to decipher the regulation of lipid accumulation in Y. lipolytica. As the transcriptional regulatory network has undergone significant evolutionary restructuring in fungi, including in the distantly related Y. lipolytica (Lavoie, Hogues and Whiteway 2009; Lavoie et al.2010; Maguire et al.2014), it would be misguided to assume that the TF regulatory network and TF functions in S. cerevisiae reflect those in other yeast. However, in Y. lipolytica, only a few TFs have been studied in the context of lipid metabolism. They include Por1, a zinc finger TF ortholog of farA in Aspergillus nidulans (Poopanitpan et al.2010); Mig1, a zinc finger TF (Wang et al.2013); the basic helix-loop-helix and Opi1-like TFs (i.e. Yas1, Yas2 and Yas3) (Endoh-Yamagami et al.2007; Hirakawa et al.2009; Kobayashi et al.2015); and, more recently, four genuine GATA-binding zinc finger (GZF) proteins and two GATA-like binding proteins (i.e. genes gzf1 to gzf6) (Pomraning, Bredeweg and Baker 2017). The putative roles of most Y. lipolytica TFs remain unknown or undescribed. There are many different classes of TFs in general and around 80 exist in fungi (Shelest 2017). It is currently estimated that the S. cerevisiae genome contains approximately 200 TF genes (Hughes and de Boer 2013). Even if S. cerevisiae is distant from Y. lipolytica, they have roughly the same number of coding sequences. We can speculate that the number of TFs in Y. lipolytica is in the same order of magnitude. Here, we developed a systematic overexpression approach in Y. lipolytica to identify putative TFs involved in lipid metabolism. We identified 148 putative TFs in the Y. lipolytica’s genome; we obtained 125 yeast transformant strains that each overexpressed a putative TF; and we found that 38 of the TFs had an impact on lipid accumulation under at least one growth condition. MATERIALS AND METHODS Identification of putative TFs All the coding sequences for Y. lipolytica were extracted from the GRYC database (http://gryc.inra.fr/). We first searched for descriptions of TFs in the annotations. We also searched for putative TF motifs using the PFAM database (http://pfam.xfam.org/); the threshold was a PFAM significance of one. To extend the list of putative TFs, known S. cerevisiae TFs were BLASTP searched against the Y. lipolytica proteome. All putative TFs previously identified in Y. lipolytica using alternative approaches were also included. The TF-specific PFAM domains that were identified in Y. lipolytica coding sequences are provided in Table S1, Supporting Information. Gateway cloning of putative TFs TFs were cloned in Gateway donor vectors and transferred to our Gateway expression vector JMP1529 (Leplat, Nicaud and Rossignol 2015) using Gateway LR clonase (Thermo Fisher Scientific, Villebon-sur-Yvette, France) in accordance with the manufacturer's instructions. JMP1529 is an integrative vector that allows, after LR recombination with donor vectors, to place genes under the control of the strong pTEF promoter. The donor vectors had one of two origins. First, some came from a sequenced cDNA library constructed with the CloneMiner cDNA Library Construction Kit (Thermo Fisher Scientific) that provides cDNA in a Gateway Entry vector (Mekouar et al.2010). Second, if they were not available from the collection, they were PCR amplified from genomic DNA and cloned using a pENTR Directional TOPO® Cloning Kit (Thermo Fisher Scientific) in accordance with the manufacturer's instructions. The origin of the TF cloning methods as well as the primer sequences for genomic DNA amplification (when applicable) are indicated in Table S1, Supporting Information. Yarrowia lipolytica transformation Yarrowia lipolytica strain JMY2566 was used in overexpression cassette transformation employing the recently developed high-throughput method for Y. lipolytica transformant library screening (Leplat, Nicaud and Rossignol 2015). When the first transformation failed, subsequent transformations were carried out using the standard lithium acetate method (Le Dall, Nicaud and Gaillardin 1994). Transformants were selected on YNB medium for URA3 complementation. Integration of the expression cassettes at the specific URA3-zeta platform in the JMY2566 genome was verified by PCR as described elsewhere (Leplat, Nicaud and Rossignol 2015). The control strain for all the experiments—JMY2810—was derived from JMY2566 by integrating an empty overexpression cassette at the URA3-zeta locus (Leplat, Nicaud and Rossignol 2015). For growth in 96 well plates, the prototroph strain JMY2900 (Dulermo et al.2014) was also used as a second control strain. Growth and media The URA + transformants were selected after incubation at 28°C on solid minimal YNB medium. YNB is composed of 0.17% (w/v) of yeast nitrogen base without ammonium and without amino acids, 0.5% (w/v) of ammonium chloride, 50 mM phosphate buffer (pH 6.8) and 1% (w/v) glucose, complemented with 1.5% (w/v) agar for solid media. Growth screening was performed using 96-well plates; 5 μL of a 24-h YPD (1% yeast extract, 1% Bacto Peptone, 2% glucose) pre-culture was used to inoculate 195 μL of YNB medium that had either 1% glucose or 1% glycerol. These compounds are the two major carbon sources used by Y. lipolytica. Cultures were grown at 28°C under constant agitation on a Biotek Synergy MX microtiter plate reader (Biotek Instruments, Colmar, France); growth was monitored by measuring optical density (OD) at 600 nm every 10 min for 24 h. Above that time in microtiter plate reader (around OD = 1.2), the correlation between OD and cell density is not linear anymore. Each treatment was performed in duplicate in separate experiments. To characterize transformant growth, maximum growth rate (Vmax, OD h−1) and maximum cell density (max OD) were calculated using a sliding eight-point scale using GEN5 2.0 software (Biotek Instruments). We considered that growth had been affected when strains displayed a mean 20% increase or decrease in Vmax or max OD relative to the median values of the six control strains present on the same plate. To determine lipid production, cultures were grown in 250 mL flasks in 25 mL YNB media containing either 3% glucose or 3% glycerol as the carbon source; the nitrogen concentration was adjusted to obtain a carbon-to-nitrogen ratio of 30 (C/N 30) for optimal lipid production (Gajdoš et al.2016). Cultures were sampled after 72 h of growth. Flask cultures were performed by batch of 20 to 30 flasks. A control strain (JMY2810) was systematically run in each batch of culture and lipids were extracted and analyzed at the same time. Samples were compared to the control strain of the same batch to avoid technical bias. Each transformant was cultured twice in separate series. Therefore, we compare percentage of variation reported to control strain used in the batch, rather than absolute data. Quantification of total lipid content Cells were harvested, washed with deionized water and freeze-dried. Fatty acids from an aliquot of lyophilized biomass (10–20 mg) were converted to their fatty acid methyl esters (FAMEs) using the Browse method (Browse, McCourt and Somerville 1986). Dried cells were mixed with 1 mL of 2.5% (v/v) sulfuric acid in methanol, which contained 50 μg of dodecanoic acid (Sigma, Saint Louis, MO, USA) as an internal standard, and were then incubated at 80°C for 90 min to form FAMEs. After transesterification, 1.5 mL of water was added, and FAMEs were extracted through the addition of 450 μL of hexane. The FAME-containing hexane phase was analyzed by gas chromatography using a Varian 3900 unit equipped with a flame ionization detector and a Varian FactorFour vf-23ms column, where the bleed specification at 260°C was 3 pA (30 m, 0.25 mm, 0.25 μm). Fatty acids were identified by comparison to commercial FAME standards (FAME32; Supelco, Sigma Aldrich, Saint-Quentin Fallavier, France) and quantified using the internal standard. RESULTS TF identification and construction of overexpression strains Few TFs have been experimentally described in Y. lipolytica. It is important to note that the definition of a TF is dependent on the exact criteria used, which leads to large discrepancies in TF identification. To compile a relatively exhaustive and highly reliable TF library before initiating cloning, several strategies were employed. First, we searched the literature and gene descriptions in genomic databases for known TFs. We then identified additional putative TFs using a PFAM domain search and a BLASTP comparison, as described in the Methods. We ended up with a list of 148 putative TFs for the Y. lipolytica genome (Table S1, Supporting Information). All 148 putative TFs were successfully cloned using our Gateway overexpression system and overexpression cassettes were used to transform the strain JMY2566. The transformation process created 125 different overexpressing strains—a cloning success rate of over 85% (i.e. from initial cloning to yeast transformation). The 23 other overexpression cassettes that failed to produce transformants were included in repeated but unsuccessful transformation procedures (5 or more repetitions). We believe that overexpression of these TFs is somehow toxic and results in transformant death. Looking for potential orthologs in S. cerevisiae does not reveal any GO enrichment neither for overepressed or knock out homologs phenotype. However, the weak similarity observed for most of these genes with S. cerevisiae orthologs do not allow to properly detect any enrichment. All transformants were tested by PCR to confirm the integration of the overexpression cassette at the URA3-zeta locus (Leplat, Nicaud and Rossignol 2015). There was a 70% rate of integration at the appropriate locus (89/125) (Table S1, Supporting Information). In the other transformants, integration occurred at random genome locations which could alter the level of expression of the overexpressed gene and may affect the detection level of possible phenotype. Overexpression has been confirmed for the mutant overexpressing TF072 (YALI0C22682g), with an increase fold change of 9 compare to control strain (Fig. S1, Supporting Information). Transformant growth As TFs can have a wide range of effects on cellular processes, we first checked our collection of TF-overexpressing strains to identify mutants with possible growth defects. To obtain precise growth kinetics, all transformants were screened on 96-well plates using glucose or glycerol in a 96-well plate reader rather than making single point measurements in flask which might only reflect maximum OD differences. Even if these growth conditions are slightly different from the flask cultures performed for lipid content measurement in term of agitation and oxygenation, it gives an overview of growth perturbation potentially generated by the gene overexpression. Based on maximum growth rate and cell density (see Methods and Table S2, Supporting Information), we identified four transformants whose growth was affected in glucose medium (i.e. decreased growth: TF072, TF079, TF099 and TF116) (Fig. 1A) and nine mutants whose growth was affected in glycerol medium (i.e. decreased growth: TF069, TF112 and TF131; increased growth: TF037, TF073, TF085, TF089, TF090 and TF091) (examples given in Fig. 1B). Most of these transformants were found to have near-threshold growth rates and cell densities on the opposing medium, indicating that for most of them, the overexpression may have a general effect on growth but is modulated by the carbon source. Figure 1. View largeDownload slide (A) Growth in glucose medium of the four mutants with glucose-growth defects (96-well plates). (B) Growth in glycerol medium of some representative mutants with glycerol-growth increase or decrease (96-well plates). Figure 1. View largeDownload slide (A) Growth in glucose medium of the four mutants with glucose-growth defects (96-well plates). (B) Growth in glycerol medium of some representative mutants with glycerol-growth increase or decrease (96-well plates). Analysis of total lipid content The ability of the TF-overexpressing strains to accumulate lipids was assessed after 72 h of growth in glucose or glycerol medium. Comparisons with the control strain allowed us to identify transformants whose lipid-accumulation phenotype was affected (Table S3, Supporting Information). In Fig. 2, we have highlighted the transformants whose lipid accumulation abilities changed the most; our threshold was a 15% increase or decrease in lipid content relative to that of the control strain (this later ranging from 8% to 10% of the dry cell weight). In glucose medium, we identified six mutants with decreased lipid content and 20 mutants with increased lipid content; the % change ranged from –26.6% (TF124; YALI0A16841g) to +90.9% (TF053; YALI0E31757g) (Fig. 2A; Table 1). In glycerol medium, we identified 13 mutants with decreased lipid content and 7 mutants with increased lipid content; the % change ranged from –55.7% (TF099; YALI0A18469g) to +74% (TF108; YALI0F25861g) (Fig. 2B; Table 1). Three transformants had increased lipid content in both media (TF019; YALI0C13178g, TF084; YALI0B08734g and TF072; YALI0C22682g), and two had decreased lipid content in both media (TF099; YALI0A18469g and TF107; YALI0C13750g). Two presented a growth defect in both glucose and glycerol media, albeit to a lesser extent in the latter case (TF072; YALI0C22682g and TF099; YALI0A18469g) (Table S2, Supporting Information). Overall, there was a weak correlation in lipid content changes in glucose versus glycerol medium for the transformants (Spearman's rank correlation: rs = 0.1539). Figure 2. View largeDownload slide Overexpressing transformants displaying notable lipid-accumulation phenotypes. (A) Percentage change in lipid content by the overexpressing strains relative to the control strain when grown in glucose medium. (B) Percentage change in lipid content by the overexpressing strains relative to the control strain when grown in glycerol medium. Figure 2. View largeDownload slide Overexpressing transformants displaying notable lipid-accumulation phenotypes. (A) Percentage change in lipid content by the overexpressing strains relative to the control strain when grown in glucose medium. (B) Percentage change in lipid content by the overexpressing strains relative to the control strain when grown in glycerol medium. Table 1. List of overexpressing transformants displaying lipid-accumulation phenotypes. Glucose Percentage change in lipid content (relative to control)a TF number ID Mean Std deviation Description Name TF124 YALI0A16841g –26.6 1.00 Conserved hypothetical protein TF099 YALI0A18469g –23.3 1.34 Homeobox protein HOY1 TF107 YALI0C13750g –20.8 1.11 Conserved hypothetical protein some similarities with S. cerevisiae YKL062W MSN4 transcriptional activator TF127 YALI0E18304g –17.5 2.01 Conserved hypothetical protein some similarities with S. cerevisiae YBR239c Putative 60.3 kDa transcriptional regulatory protein TF112 YALI0C19063g –16.9 0.68 Conserved hypothetical protein weakly similar to Nectria haematococca hypothetical protein NECHADRAFT_43519 TF051 YALI0E31383g –15.5 0.85 Conserved hypothetical protein weakly similar to Neurospora crassa hypothetical protein TF046 YALI0E17721g 16.7 0.92 Conserved hypothetical protein similar to Spathaspora passalidarum SPAPADRAFT_137815 hypothetical protein TF003 YALI0B00660g 18.8 0.64 Conserved hypothetical protein weakly similar to S. cerevisiae YDR213W UPC2 regulatory protein involved in control of sterol uptake TF059 YALI0F06072g 19.2 0.85 Conserved hypothetical protein some similarities with N. crassa Probable cutinase transcription factor 1 beta TF071 YALI0E10131g 20.8 3.54 Transcription elongation factor with a conserved zinc finger domain, similar to S. cerevisiae YKL160W ELF1 Transcription elongation factor with a conserved zinc finger domain TF072 YALI0C22682g 21.0 0.57 Conserved hypothetical protein some similarities with S. cerevisiae YJL110C GZF3 transcriptional repressor GZF3 TF074 YALI0D09757g 21.9 0.14 Conserved hypothetical protein weakly similar to S. cerevisiae YHL009c YAP3 transcription factor, of a fungal-specific family of bzip proteins TF128 YALI0F05896g 22.5 1.27 Conserved hypothetical protein weakly similar to Tuber melanosporum hypothetical protein TF040 YALI0E03410g 22.6 0.76 Conserved hypothetical protein weakly similar to S. cerevisiae YBR239c TF085 YALI0C11858g 28.0 0.92 Gal4-type transcription factor, weakly similar to Y. lipolytica YALI1E31383g, putative sequence-specific DNA binding RNA polymerase II transcription factor TF139 Yali0E05577g 28.3 1.48 Hypothetical protein of a 6-member family, conserved in the Yarrowia clade highly similar to Y. lipolytica YALI1F13761g, protein with HXXEE motif TF055 YALI0F03157g 32.6 0.92 Conserved hypothetical protein some similarities with S. cerevisiae YDR253C MET32 transcriptional regulator of sulfur amino acid metabolism TF135 Yali0E14971g 35.6 4.88 Conserved hypothetical protein some similarities with S. cerevisiae YGR044c Zinc finger protein RME1 and other transcription factors TF109 YALI0E16577g 37.2 4.31 Conserved hypothetical protein Forward some similarities with S. cerevisiae YKL185w ASH1 negative regulator of HO transcription GZF5 TF019 YALI0C13178g 41.0 3.54 Conserved hypothetical protein some similarities with S. cerevisiae YOR344c TYE7 basic helix-loop-helix transcription factor TF057 YALI0F05104g 43.2 6.72 Conserved hypothetical protein weakly similar to S. cerevisiae YPR186c TFC2 TFIIIA transcription initiation factor TF084 YALI0B08734g 50.5 3.25 Cytoplasmic pre-60S factor, putative similar to S. cerevisiae YBR267W REI1 Cytoplasmic pre-60S factor TF064 YALI0F16599g 53.5 1.13 Conserved hypothetical protein weakly similar to Debaryomyces hansenii DEHA2B03564p TF086 YALI0B04510g 57.2 2.97 Conserved hypothetical weakly similar to S. cerevisiae YKL032C IXR1 intrastrand crosslink recognition protein and transcription factor TF077 YALI0B08206g 85.7 1.98 Y. lipolytica YALI1B08206g CRF1 Copper resistance protein CRF1 TF053 YALI0E31757g 90.9 6.15 Conserved hypothetical protein weakly similar to N. crassa 123A4.250 Related to NsdD protein TF099 YALI0A18469g –55.75 24.40 Homeobox protein HOY1 TF107 YALI0C13750g –41.65 1.63 Conserved hypothetical protein some similarities with S. cerevisiae YKL062W MSN4 transcriptional activator TF089 YALI0C06842g –35.2 11.31 Transcription factor, putative similar to S. cerevisiae YMR043W MCM1 Transcription factor involved in cell-type-specific transcription and pheromone response TF073 YALI0B15818g –34.8 3.25 Conserved hypothetical protein some similarities with S. cerevisiae YDR213W UPC2 Sterol regulatory element binding protein TF050 YALI0E30789g –31.25 3.75 Conserved hypothetical protein weakly similar to Ajellomyces dermatitidis C2H2 finger domain-containing protein TF083 YALI0C02387g –31 1.56 Y. lipolytica YALI1C02387g YAS1 Basic helix-loop-helix transcription factor essential for cytochrome p450 induction in response to alkanes YAS1 TF068 YALI0E13948g –28.65 4.88 Conserved hypothetical protein some similarities with S. cerevisiae YGL073w HSF1 heat shock transcription factor TF076 YALI0D02475g –27.3 1.27 Conserved hypothetical protein weakly similar to Sclerotinia sclerotiorum hypothetical protein SS1G_03399 TF109 YALI0E16577g –23.45 3.89 Conserved hypothetical protein some similarities with S. cerevisiae YKL185w ASH1 negative regulator of HO transcription GZF5 TF098 YALI0D01463g –22.2 1.56 Conserved hypothetical protein some similarities with S. cerevisiae YNL027w CRZ1 calcineurin responsive zinc finger TF064 YALI0F16599g –20.7 3.68 Conserved hypothetical protein weakly similar to D. hansenii DEHA2B03564p TF075 YALI0F05346g –16.1 0.14 Conserved hypothetical protein weakly similar to Fusarium solani Cutinase gene palindrome-binding protein TF077 YALI0B08206g –16.05 0.07 Y. lipolytica YALI1B08206g CRF1 Copper resistance protein CRF1 TF039 YALI0D23749g 17.95 1.63 Conserved hypothetical protein weakly similar to S. cerevisiae YJL056c ZAP1 metalloregulatory protein involved in zinc-responsive transcriptional regulation TF079 YALI0D12628g 18.05 0.07 Y. lipolytica YALI1D12628g POR1 Subunit of the SWI/SNF chromatin remodeling complex involved in transcriptional regulation POR1 TF019 YALI0C13178g 18.3 0.57 Conserved hypothetical protein some similarities with S. cerevisiae YOR344c TYE7 basic helix-loop-helix transcription factor TF084 YALI0B08734g 18.45 0.07 Cytoplasmic pre-60S factor, similar to S. cerevisiae YBR267W REI1 Cytoplasmic pre-60S factor TF072 YALI0C22682g 22.4 4.53 Conserved hypothetical protein some similarities with S. cerevisiae YJL110C GZF3 transcriptional repressor GZF3 TF001 YALI0A10637g 27.3 1.13 Conserved hypothetical protein TF108 YALI0F25861g 74 22.34 Conserved hypothetical protein weakly similar to S. cerevisiae YDL020c SON1 26S proteasome subunit Glucose Percentage change in lipid content (relative to control)a TF number ID Mean Std deviation Description Name TF124 YALI0A16841g –26.6 1.00 Conserved hypothetical protein TF099 YALI0A18469g –23.3 1.34 Homeobox protein HOY1 TF107 YALI0C13750g –20.8 1.11 Conserved hypothetical protein some similarities with S. cerevisiae YKL062W MSN4 transcriptional activator TF127 YALI0E18304g –17.5 2.01 Conserved hypothetical protein some similarities with S. cerevisiae YBR239c Putative 60.3 kDa transcriptional regulatory protein TF112 YALI0C19063g –16.9 0.68 Conserved hypothetical protein weakly similar to Nectria haematococca hypothetical protein NECHADRAFT_43519 TF051 YALI0E31383g –15.5 0.85 Conserved hypothetical protein weakly similar to Neurospora crassa hypothetical protein TF046 YALI0E17721g 16.7 0.92 Conserved hypothetical protein similar to Spathaspora passalidarum SPAPADRAFT_137815 hypothetical protein TF003 YALI0B00660g 18.8 0.64 Conserved hypothetical protein weakly similar to S. cerevisiae YDR213W UPC2 regulatory protein involved in control of sterol uptake TF059 YALI0F06072g 19.2 0.85 Conserved hypothetical protein some similarities with N. crassa Probable cutinase transcription factor 1 beta TF071 YALI0E10131g 20.8 3.54 Transcription elongation factor with a conserved zinc finger domain, similar to S. cerevisiae YKL160W ELF1 Transcription elongation factor with a conserved zinc finger domain TF072 YALI0C22682g 21.0 0.57 Conserved hypothetical protein some similarities with S. cerevisiae YJL110C GZF3 transcriptional repressor GZF3 TF074 YALI0D09757g 21.9 0.14 Conserved hypothetical protein weakly similar to S. cerevisiae YHL009c YAP3 transcription factor, of a fungal-specific family of bzip proteins TF128 YALI0F05896g 22.5 1.27 Conserved hypothetical protein weakly similar to Tuber melanosporum hypothetical protein TF040 YALI0E03410g 22.6 0.76 Conserved hypothetical protein weakly similar to S. cerevisiae YBR239c TF085 YALI0C11858g 28.0 0.92 Gal4-type transcription factor, weakly similar to Y. lipolytica YALI1E31383g, putative sequence-specific DNA binding RNA polymerase II transcription factor TF139 Yali0E05577g 28.3 1.48 Hypothetical protein of a 6-member family, conserved in the Yarrowia clade highly similar to Y. lipolytica YALI1F13761g, protein with HXXEE motif TF055 YALI0F03157g 32.6 0.92 Conserved hypothetical protein some similarities with S. cerevisiae YDR253C MET32 transcriptional regulator of sulfur amino acid metabolism TF135 Yali0E14971g 35.6 4.88 Conserved hypothetical protein some similarities with S. cerevisiae YGR044c Zinc finger protein RME1 and other transcription factors TF109 YALI0E16577g 37.2 4.31 Conserved hypothetical protein Forward some similarities with S. cerevisiae YKL185w ASH1 negative regulator of HO transcription GZF5 TF019 YALI0C13178g 41.0 3.54 Conserved hypothetical protein some similarities with S. cerevisiae YOR344c TYE7 basic helix-loop-helix transcription factor TF057 YALI0F05104g 43.2 6.72 Conserved hypothetical protein weakly similar to S. cerevisiae YPR186c TFC2 TFIIIA transcription initiation factor TF084 YALI0B08734g 50.5 3.25 Cytoplasmic pre-60S factor, putative similar to S. cerevisiae YBR267W REI1 Cytoplasmic pre-60S factor TF064 YALI0F16599g 53.5 1.13 Conserved hypothetical protein weakly similar to Debaryomyces hansenii DEHA2B03564p TF086 YALI0B04510g 57.2 2.97 Conserved hypothetical weakly similar to S. cerevisiae YKL032C IXR1 intrastrand crosslink recognition protein and transcription factor TF077 YALI0B08206g 85.7 1.98 Y. lipolytica YALI1B08206g CRF1 Copper resistance protein CRF1 TF053 YALI0E31757g 90.9 6.15 Conserved hypothetical protein weakly similar to N. crassa 123A4.250 Related to NsdD protein TF099 YALI0A18469g –55.75 24.40 Homeobox protein HOY1 TF107 YALI0C13750g –41.65 1.63 Conserved hypothetical protein some similarities with S. cerevisiae YKL062W MSN4 transcriptional activator TF089 YALI0C06842g –35.2 11.31 Transcription factor, putative similar to S. cerevisiae YMR043W MCM1 Transcription factor involved in cell-type-specific transcription and pheromone response TF073 YALI0B15818g –34.8 3.25 Conserved hypothetical protein some similarities with S. cerevisiae YDR213W UPC2 Sterol regulatory element binding protein TF050 YALI0E30789g –31.25 3.75 Conserved hypothetical protein weakly similar to Ajellomyces dermatitidis C2H2 finger domain-containing protein TF083 YALI0C02387g –31 1.56 Y. lipolytica YALI1C02387g YAS1 Basic helix-loop-helix transcription factor essential for cytochrome p450 induction in response to alkanes YAS1 TF068 YALI0E13948g –28.65 4.88 Conserved hypothetical protein some similarities with S. cerevisiae YGL073w HSF1 heat shock transcription factor TF076 YALI0D02475g –27.3 1.27 Conserved hypothetical protein weakly similar to Sclerotinia sclerotiorum hypothetical protein SS1G_03399 TF109 YALI0E16577g –23.45 3.89 Conserved hypothetical protein some similarities with S. cerevisiae YKL185w ASH1 negative regulator of HO transcription GZF5 TF098 YALI0D01463g –22.2 1.56 Conserved hypothetical protein some similarities with S. cerevisiae YNL027w CRZ1 calcineurin responsive zinc finger TF064 YALI0F16599g –20.7 3.68 Conserved hypothetical protein weakly similar to D. hansenii DEHA2B03564p TF075 YALI0F05346g –16.1 0.14 Conserved hypothetical protein weakly similar to Fusarium solani Cutinase gene palindrome-binding protein TF077 YALI0B08206g –16.05 0.07 Y. lipolytica YALI1B08206g CRF1 Copper resistance protein CRF1 TF039 YALI0D23749g 17.95 1.63 Conserved hypothetical protein weakly similar to S. cerevisiae YJL056c ZAP1 metalloregulatory protein involved in zinc-responsive transcriptional regulation TF079 YALI0D12628g 18.05 0.07 Y. lipolytica YALI1D12628g POR1 Subunit of the SWI/SNF chromatin remodeling complex involved in transcriptional regulation POR1 TF019 YALI0C13178g 18.3 0.57 Conserved hypothetical protein some similarities with S. cerevisiae YOR344c TYE7 basic helix-loop-helix transcription factor TF084 YALI0B08734g 18.45 0.07 Cytoplasmic pre-60S factor, similar to S. cerevisiae YBR267W REI1 Cytoplasmic pre-60S factor TF072 YALI0C22682g 22.4 4.53 Conserved hypothetical protein some similarities with S. cerevisiae YJL110C GZF3 transcriptional repressor GZF3 TF001 YALI0A10637g 27.3 1.13 Conserved hypothetical protein TF108 YALI0F25861g 74 22.34 Conserved hypothetical protein weakly similar to S. cerevisiae YDL020c SON1 26S proteasome subunit aThe strains that showed notably decreased and increased lipid contents are in green and red, respectively. View Large Table 1. List of overexpressing transformants displaying lipid-accumulation phenotypes. Glucose Percentage change in lipid content (relative to control)a TF number ID Mean Std deviation Description Name TF124 YALI0A16841g –26.6 1.00 Conserved hypothetical protein TF099 YALI0A18469g –23.3 1.34 Homeobox protein HOY1 TF107 YALI0C13750g –20.8 1.11 Conserved hypothetical protein some similarities with S. cerevisiae YKL062W MSN4 transcriptional activator TF127 YALI0E18304g –17.5 2.01 Conserved hypothetical protein some similarities with S. cerevisiae YBR239c Putative 60.3 kDa transcriptional regulatory protein TF112 YALI0C19063g –16.9 0.68 Conserved hypothetical protein weakly similar to Nectria haematococca hypothetical protein NECHADRAFT_43519 TF051 YALI0E31383g –15.5 0.85 Conserved hypothetical protein weakly similar to Neurospora crassa hypothetical protein TF046 YALI0E17721g 16.7 0.92 Conserved hypothetical protein similar to Spathaspora passalidarum SPAPADRAFT_137815 hypothetical protein TF003 YALI0B00660g 18.8 0.64 Conserved hypothetical protein weakly similar to S. cerevisiae YDR213W UPC2 regulatory protein involved in control of sterol uptake TF059 YALI0F06072g 19.2 0.85 Conserved hypothetical protein some similarities with N. crassa Probable cutinase transcription factor 1 beta TF071 YALI0E10131g 20.8 3.54 Transcription elongation factor with a conserved zinc finger domain, similar to S. cerevisiae YKL160W ELF1 Transcription elongation factor with a conserved zinc finger domain TF072 YALI0C22682g 21.0 0.57 Conserved hypothetical protein some similarities with S. cerevisiae YJL110C GZF3 transcriptional repressor GZF3 TF074 YALI0D09757g 21.9 0.14 Conserved hypothetical protein weakly similar to S. cerevisiae YHL009c YAP3 transcription factor, of a fungal-specific family of bzip proteins TF128 YALI0F05896g 22.5 1.27 Conserved hypothetical protein weakly similar to Tuber melanosporum hypothetical protein TF040 YALI0E03410g 22.6 0.76 Conserved hypothetical protein weakly similar to S. cerevisiae YBR239c TF085 YALI0C11858g 28.0 0.92 Gal4-type transcription factor, weakly similar to Y. lipolytica YALI1E31383g, putative sequence-specific DNA binding RNA polymerase II transcription factor TF139 Yali0E05577g 28.3 1.48 Hypothetical protein of a 6-member family, conserved in the Yarrowia clade highly similar to Y. lipolytica YALI1F13761g, protein with HXXEE motif TF055 YALI0F03157g 32.6 0.92 Conserved hypothetical protein some similarities with S. cerevisiae YDR253C MET32 transcriptional regulator of sulfur amino acid metabolism TF135 Yali0E14971g 35.6 4.88 Conserved hypothetical protein some similarities with S. cerevisiae YGR044c Zinc finger protein RME1 and other transcription factors TF109 YALI0E16577g 37.2 4.31 Conserved hypothetical protein Forward some similarities with S. cerevisiae YKL185w ASH1 negative regulator of HO transcription GZF5 TF019 YALI0C13178g 41.0 3.54 Conserved hypothetical protein some similarities with S. cerevisiae YOR344c TYE7 basic helix-loop-helix transcription factor TF057 YALI0F05104g 43.2 6.72 Conserved hypothetical protein weakly similar to S. cerevisiae YPR186c TFC2 TFIIIA transcription initiation factor TF084 YALI0B08734g 50.5 3.25 Cytoplasmic pre-60S factor, putative similar to S. cerevisiae YBR267W REI1 Cytoplasmic pre-60S factor TF064 YALI0F16599g 53.5 1.13 Conserved hypothetical protein weakly similar to Debaryomyces hansenii DEHA2B03564p TF086 YALI0B04510g 57.2 2.97 Conserved hypothetical weakly similar to S. cerevisiae YKL032C IXR1 intrastrand crosslink recognition protein and transcription factor TF077 YALI0B08206g 85.7 1.98 Y. lipolytica YALI1B08206g CRF1 Copper resistance protein CRF1 TF053 YALI0E31757g 90.9 6.15 Conserved hypothetical protein weakly similar to N. crassa 123A4.250 Related to NsdD protein TF099 YALI0A18469g –55.75 24.40 Homeobox protein HOY1 TF107 YALI0C13750g –41.65 1.63 Conserved hypothetical protein some similarities with S. cerevisiae YKL062W MSN4 transcriptional activator TF089 YALI0C06842g –35.2 11.31 Transcription factor, putative similar to S. cerevisiae YMR043W MCM1 Transcription factor involved in cell-type-specific transcription and pheromone response TF073 YALI0B15818g –34.8 3.25 Conserved hypothetical protein some similarities with S. cerevisiae YDR213W UPC2 Sterol regulatory element binding protein TF050 YALI0E30789g –31.25 3.75 Conserved hypothetical protein weakly similar to Ajellomyces dermatitidis C2H2 finger domain-containing protein TF083 YALI0C02387g –31 1.56 Y. lipolytica YALI1C02387g YAS1 Basic helix-loop-helix transcription factor essential for cytochrome p450 induction in response to alkanes YAS1 TF068 YALI0E13948g –28.65 4.88 Conserved hypothetical protein some similarities with S. cerevisiae YGL073w HSF1 heat shock transcription factor TF076 YALI0D02475g –27.3 1.27 Conserved hypothetical protein weakly similar to Sclerotinia sclerotiorum hypothetical protein SS1G_03399 TF109 YALI0E16577g –23.45 3.89 Conserved hypothetical protein some similarities with S. cerevisiae YKL185w ASH1 negative regulator of HO transcription GZF5 TF098 YALI0D01463g –22.2 1.56 Conserved hypothetical protein some similarities with S. cerevisiae YNL027w CRZ1 calcineurin responsive zinc finger TF064 YALI0F16599g –20.7 3.68 Conserved hypothetical protein weakly similar to D. hansenii DEHA2B03564p TF075 YALI0F05346g –16.1 0.14 Conserved hypothetical protein weakly similar to Fusarium solani Cutinase gene palindrome-binding protein TF077 YALI0B08206g –16.05 0.07 Y. lipolytica YALI1B08206g CRF1 Copper resistance protein CRF1 TF039 YALI0D23749g 17.95 1.63 Conserved hypothetical protein weakly similar to S. cerevisiae YJL056c ZAP1 metalloregulatory protein involved in zinc-responsive transcriptional regulation TF079 YALI0D12628g 18.05 0.07 Y. lipolytica YALI1D12628g POR1 Subunit of the SWI/SNF chromatin remodeling complex involved in transcriptional regulation POR1 TF019 YALI0C13178g 18.3 0.57 Conserved hypothetical protein some similarities with S. cerevisiae YOR344c TYE7 basic helix-loop-helix transcription factor TF084 YALI0B08734g 18.45 0.07 Cytoplasmic pre-60S factor, similar to S. cerevisiae YBR267W REI1 Cytoplasmic pre-60S factor TF072 YALI0C22682g 22.4 4.53 Conserved hypothetical protein some similarities with S. cerevisiae YJL110C GZF3 transcriptional repressor GZF3 TF001 YALI0A10637g 27.3 1.13 Conserved hypothetical protein TF108 YALI0F25861g 74 22.34 Conserved hypothetical protein weakly similar to S. cerevisiae YDL020c SON1 26S proteasome subunit Glucose Percentage change in lipid content (relative to control)a TF number ID Mean Std deviation Description Name TF124 YALI0A16841g –26.6 1.00 Conserved hypothetical protein TF099 YALI0A18469g –23.3 1.34 Homeobox protein HOY1 TF107 YALI0C13750g –20.8 1.11 Conserved hypothetical protein some similarities with S. cerevisiae YKL062W MSN4 transcriptional activator TF127 YALI0E18304g –17.5 2.01 Conserved hypothetical protein some similarities with S. cerevisiae YBR239c Putative 60.3 kDa transcriptional regulatory protein TF112 YALI0C19063g –16.9 0.68 Conserved hypothetical protein weakly similar to Nectria haematococca hypothetical protein NECHADRAFT_43519 TF051 YALI0E31383g –15.5 0.85 Conserved hypothetical protein weakly similar to Neurospora crassa hypothetical protein TF046 YALI0E17721g 16.7 0.92 Conserved hypothetical protein similar to Spathaspora passalidarum SPAPADRAFT_137815 hypothetical protein TF003 YALI0B00660g 18.8 0.64 Conserved hypothetical protein weakly similar to S. cerevisiae YDR213W UPC2 regulatory protein involved in control of sterol uptake TF059 YALI0F06072g 19.2 0.85 Conserved hypothetical protein some similarities with N. crassa Probable cutinase transcription factor 1 beta TF071 YALI0E10131g 20.8 3.54 Transcription elongation factor with a conserved zinc finger domain, similar to S. cerevisiae YKL160W ELF1 Transcription elongation factor with a conserved zinc finger domain TF072 YALI0C22682g 21.0 0.57 Conserved hypothetical protein some similarities with S. cerevisiae YJL110C GZF3 transcriptional repressor GZF3 TF074 YALI0D09757g 21.9 0.14 Conserved hypothetical protein weakly similar to S. cerevisiae YHL009c YAP3 transcription factor, of a fungal-specific family of bzip proteins TF128 YALI0F05896g 22.5 1.27 Conserved hypothetical protein weakly similar to Tuber melanosporum hypothetical protein TF040 YALI0E03410g 22.6 0.76 Conserved hypothetical protein weakly similar to S. cerevisiae YBR239c TF085 YALI0C11858g 28.0 0.92 Gal4-type transcription factor, weakly similar to Y. lipolytica YALI1E31383g, putative sequence-specific DNA binding RNA polymerase II transcription factor TF139 Yali0E05577g 28.3 1.48 Hypothetical protein of a 6-member family, conserved in the Yarrowia clade highly similar to Y. lipolytica YALI1F13761g, protein with HXXEE motif TF055 YALI0F03157g 32.6 0.92 Conserved hypothetical protein some similarities with S. cerevisiae YDR253C MET32 transcriptional regulator of sulfur amino acid metabolism TF135 Yali0E14971g 35.6 4.88 Conserved hypothetical protein some similarities with S. cerevisiae YGR044c Zinc finger protein RME1 and other transcription factors TF109 YALI0E16577g 37.2 4.31 Conserved hypothetical protein Forward some similarities with S. cerevisiae YKL185w ASH1 negative regulator of HO transcription GZF5 TF019 YALI0C13178g 41.0 3.54 Conserved hypothetical protein some similarities with S. cerevisiae YOR344c TYE7 basic helix-loop-helix transcription factor TF057 YALI0F05104g 43.2 6.72 Conserved hypothetical protein weakly similar to S. cerevisiae YPR186c TFC2 TFIIIA transcription initiation factor TF084 YALI0B08734g 50.5 3.25 Cytoplasmic pre-60S factor, putative similar to S. cerevisiae YBR267W REI1 Cytoplasmic pre-60S factor TF064 YALI0F16599g 53.5 1.13 Conserved hypothetical protein weakly similar to Debaryomyces hansenii DEHA2B03564p TF086 YALI0B04510g 57.2 2.97 Conserved hypothetical weakly similar to S. cerevisiae YKL032C IXR1 intrastrand crosslink recognition protein and transcription factor TF077 YALI0B08206g 85.7 1.98 Y. lipolytica YALI1B08206g CRF1 Copper resistance protein CRF1 TF053 YALI0E31757g 90.9 6.15 Conserved hypothetical protein weakly similar to N. crassa 123A4.250 Related to NsdD protein TF099 YALI0A18469g –55.75 24.40 Homeobox protein HOY1 TF107 YALI0C13750g –41.65 1.63 Conserved hypothetical protein some similarities with S. cerevisiae YKL062W MSN4 transcriptional activator TF089 YALI0C06842g –35.2 11.31 Transcription factor, putative similar to S. cerevisiae YMR043W MCM1 Transcription factor involved in cell-type-specific transcription and pheromone response TF073 YALI0B15818g –34.8 3.25 Conserved hypothetical protein some similarities with S. cerevisiae YDR213W UPC2 Sterol regulatory element binding protein TF050 YALI0E30789g –31.25 3.75 Conserved hypothetical protein weakly similar to Ajellomyces dermatitidis C2H2 finger domain-containing protein TF083 YALI0C02387g –31 1.56 Y. lipolytica YALI1C02387g YAS1 Basic helix-loop-helix transcription factor essential for cytochrome p450 induction in response to alkanes YAS1 TF068 YALI0E13948g –28.65 4.88 Conserved hypothetical protein some similarities with S. cerevisiae YGL073w HSF1 heat shock transcription factor TF076 YALI0D02475g –27.3 1.27 Conserved hypothetical protein weakly similar to Sclerotinia sclerotiorum hypothetical protein SS1G_03399 TF109 YALI0E16577g –23.45 3.89 Conserved hypothetical protein some similarities with S. cerevisiae YKL185w ASH1 negative regulator of HO transcription GZF5 TF098 YALI0D01463g –22.2 1.56 Conserved hypothetical protein some similarities with S. cerevisiae YNL027w CRZ1 calcineurin responsive zinc finger TF064 YALI0F16599g –20.7 3.68 Conserved hypothetical protein weakly similar to D. hansenii DEHA2B03564p TF075 YALI0F05346g –16.1 0.14 Conserved hypothetical protein weakly similar to Fusarium solani Cutinase gene palindrome-binding protein TF077 YALI0B08206g –16.05 0.07 Y. lipolytica YALI1B08206g CRF1 Copper resistance protein CRF1 TF039 YALI0D23749g 17.95 1.63 Conserved hypothetical protein weakly similar to S. cerevisiae YJL056c ZAP1 metalloregulatory protein involved in zinc-responsive transcriptional regulation TF079 YALI0D12628g 18.05 0.07 Y. lipolytica YALI1D12628g POR1 Subunit of the SWI/SNF chromatin remodeling complex involved in transcriptional regulation POR1 TF019 YALI0C13178g 18.3 0.57 Conserved hypothetical protein some similarities with S. cerevisiae YOR344c TYE7 basic helix-loop-helix transcription factor TF084 YALI0B08734g 18.45 0.07 Cytoplasmic pre-60S factor, similar to S. cerevisiae YBR267W REI1 Cytoplasmic pre-60S factor TF072 YALI0C22682g 22.4 4.53 Conserved hypothetical protein some similarities with S. cerevisiae YJL110C GZF3 transcriptional repressor GZF3 TF001 YALI0A10637g 27.3 1.13 Conserved hypothetical protein TF108 YALI0F25861g 74 22.34 Conserved hypothetical protein weakly similar to S. cerevisiae YDL020c SON1 26S proteasome subunit aThe strains that showed notably decreased and increased lipid contents are in green and red, respectively. View Large DISCUSSION In this work, we setup a relatively high-throughput overexpression strategy in the yeast Y. lipolytica in order to screen for potential phenotype. While we decided to screen all proteins with a potential TF domain as they are ideal targets for a large-scale overexpression screening, our goal was not to provide evidence for TF activities but rather identification of new genes involved directly or indirectly in lipid accumulation. Nevertheless, several TFs that we targeted here have been previously studied. Historically, the first TFs studied in Y. lipolytica were related to filamentation. TF099 (YALI0A18469g) has been described as a Hoy1 homeobox protein and is required for hyphal formation in Y. lipolytica (Torres-Guzman and Dominguez 1997): a deletion of hoy1 suppresses the filamentation phenotype. When we overexpressed Hoy1, we observed a growth defect under different conditions. Microscopic observations revealed that Hoy1 overexpression induced a strong and constitutive filamentation phenotype in various media and resulted in strains with greatly reduced viability (Fig. S2, Supporting Information). This finding supports the validity of our high-throughput overexpression screening approach. Moreover, TF099 (Hoy1) was the transformant with the most pronounced decrease in lipid content in both media, which is likely linked to the filamentation phenotype. Filamentation potentially drives the remobilization of triacylglycerol in lipid droplets (Gajdoš et al.2016). To a lesser extent, MHY1 overexpression (as seen in TF095; YALI0B21582g) also resulted in a hyperfilamentous phenotype (Fig. S2, Supporting Information); Mhy1 is a C2H2-type zinc finger protein required for dimorphic transition (Hurtado and Rachubinski 1999). Only a small decrease in lipid content resulted from MHY1 overexpression (Table S3, Supporting Information). In the literature, several TFs have been reported to be involved in lipid accumulation regulation. An ortholog of farA in A. nidulans with a zinc finger domain and a fungus-specific TF domain, Por1 appears to be a transcriptional activator that regulates fatty acid metabolism in Y. lipolytica (Poopanitpan et al.2010). In the same study, three other TFs putatively involved in lipid utilization were disrupted—YALI0F13321g, YALI0D17988g and YALI0D10681g—without obtaining notable phenotypes. Our study provided confirmation of these prior results. POR1 overexpression (TF079; YALI0D12628g) supported this TF’s role in lipid accumulation, at least when yeast were grown in glycerol medium (Table 1), and the transformants overexpressing YALI0F13321g, YALI0D17988g and YALI0D10681g did not display any lipid-accumulation phenotypes (Table S3, Supporting Information). Deletion of MIG1, a zinc finger TF, has been shown to induce lipid accumulation in Y. lipolytica (Wang et al.2013). In the corresponding transformant, genes involved in lipid metabolism were derepressed, which fits with the lipid-accumulation phenotype. However, genes involved in lipid degradation were also expressed at higher levels compared to the wild type (Wang et al.2013), indicating that regulation is complex and likely operates during translation or post-translation. Accordingly, the overexpression of MIG1 (TF042; YALI0E07942g) did not affect lipid content in our screen (Table S3, Supporting Information). Yas1 and Yas2 are two basic helix-loop-helix TFs that form a heterodimer that induces ALK1, a gene involved in alkane oxidation (Endoh-Yamagami et al.2007), while Yas3, another TF, has a repressor effect (Hirakawa et al.2009). We obtained a Yas1 overexpressing mutant (TF083; YALI0C02387g) and a mutant with a spliced form of Yas3 (TF130; YALI0C14784g). Overexpression of Yas1 triggered a decrease in lipid content (Table 1), while overexpression of Yas3 did not significantly affect lipid content, suggesting they play different roles in lipid accumulation just as they do in alkane metabolism. Recently, all putative Gzf TFs were identified in Y. lipolytica. Deletion transformants were functionally characterized to determine the TFs’ roles in nitrogen catabolic repression and lipid metabolism (Pomraning, Bredeweg and Baker 2017). We also identified these six putative Gzf TFs: TF037 (Gzf1; Yali0D20482g), TF082 (Gzf2; Yali0F17886g), TF072 (Gzf3; Yali0C22682g), TF041 (Gzf4; Yali0E05555g), TF109 (Gzf5; Yali0E16577g) and TF075 (Gzf6; Yali0E05346g). We obtained overexpression transformants for five of them (TF037, TF072, TF041, TF109 and TF075). In a previous study, Δgzf2 and Δgzf3 generated higher concentrations of lipids when ammonium sulfate was the sole nitrogen source, which was not the case when peptone plus yeast extract was used (Pomraning, Bredeweg and Baker 2017). While we did not obtain a GZF2 overexpression transformant, we were able to create a GZF3-overexpressing strain (TF072), which displayed increased lipid production in both glucose and glycerol media (Fig. 2A and B; Table 1) and also had a growth defect (Fig. 1). This increase in lipid content was somewhat surprising, although we did use a different nitrogen source (i.e. ammonium chloride), which might have affected regulation via Gzf3 since regulation is nitrogen source dependent (Pomraning, Bredeweg and Baker 2017). While the deletion of GZF5 seemed to have no effect on lipid accumulation (Pomraning, Bredeweg and Baker 2017), we found here that the GZF5-overexpression mutant had increased lipid content when grown in glucose medium; the opposite was observed in glycerol medium (Fig. 2A and B; Table 1). It is one of the three transformants that displayed opposite phenotypes depending on the carbon source (Fig. 2A and B; Table 1). These results reveal that the regulation of lipid metabolism by Gzf TFs is complex. Recently, Mga2 was found to be involved in lipid accumulation (Liu et al.2015). Mga2 is not annotated as a TF and does not present a classical TF motif (but rather an immunoglobulin-like fold). It was therefore not included in the list used to create our overexpressing strain collection. Three of the indentified TFs examined here with an effect on lipid accumulation (Hoy1, Gzf3, TF057) have been described among the 10 most influential TFs during lipid accumulation by inference of coregulatory network (Trébulle et al.2017) supporting their role in this phenotype and consolidate the relevance of the results obtained in this high throughput screening. Overall, we identified 38 overexpressing strains that had altered lipid-accumulation phenotypes under at least one condition. Surprisingly, we did not observe any significant variation in term of fatty acid profiles compare to the control. Most of the putative TFs involved had not been described in the literature as affecting lipid metabolism regulation and thus their functions had not been experimentally annotated. The paucity of biological investigation for these genes makes the determination of their exact role and function very speculative at this stage. By using two different growth media in our screen, we discovered that the regulation network probably differs depending on the carbon source because a large number of the overexpressing mutants had different phenotypes in glucose versus glycerol medium. It is well established that condition-dependent changes in the binding landscape of TFs occur in S. cerevisiae, phenomena that have been highlighted by large-scale chIP-chip analyses (see Hughes and de Boer 2013 for a review). This fact indicates that complex regulatory networks can be significantly rewired based on culturing conditions. The situation in Y. lipolytica is likely similar, as would suggest the weak correlation in phenotypes observed in the two growth media. Such information is essential to have when carrying out industrial bioprocesses and should be incorporated into synthetic biology approaches in Y. lipolytica, particularly those involving biolipid production. Therefore, we need a more detailed understanding of network rewiring and genome-scale analyses should be accelerated. To our knowledge, our study is the first systematic high-throughput functional analysis of a category of genes in Y. lipolytica. We have provided information on the putative roles of an important subset of genes with up to now unknown functions. We have also built the largest collection of Y. lipolytica overexpressing strains to date, which could also be screened for other phenotypes. SUPPLEMENTARY DATA Supplementary data are available at FEMSYR online. Acknowledgements We would like to thank Heber Gamboa-Meléndez for providing the qRT-PCR data. FUNDING This work was supported by the SAS PIVERT, within the frame of the French Institute for Energy Transition (Institut pour la Transition Energétique (ITE) P.I.V.E.R.T. (www.institut-pivert.com) selected as an Investment for the Future (‘Investissements d’Avenir’)). 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FEMS Yeast ResearchOxford University Press

Published: Mar 29, 2018

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