The plasma membrane H+-ATPase promoter enables highly efficient production of punicic acid in Rhodotorula toruloides cultivated on glucose and crude glycerol

The plasma membrane H+-ATPase promoter enables highly efficient production of punicic acid in Rhodotorula toruloides cultivated on glucose and crude glycerol | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The plasma membrane H+-ATPase promoter enables highly efficient production of punicic acid in Rhodotorula toruloides cultivated on glucose and crude glycerol Daniela Krajciova, Roman Holic This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4774339/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Punicic acid is a conjugated fatty acid with a wide-range of nutraceutical properties naturally present in pomegranate seed oil. To meet the rising demand for pomegranate seed oil, a single-cell oil enriched in punicic acid provides a sustainable biomass-derived alternative. This study describes the production of a punicic acid-enriched single-cell oil through the engineering of the red yeast Rhodotorula toruloides grown in glucose and a low-cost substrate, crude glycerol. Results The gene for Punica granatum fatty acid conjugase, PgFADX , was randomly integrated into the genome of R. toruloides without disrupting the carotenoid synthesis. In shake flask studies, the effects of three promoters (P PGI1 , P NAR1 , and P PMA1 ) on punicic acid production were evaluated. A punicic acid titer of 105.77 mg/L and 72.81 mg/L was obtained from engineered cells expressing PgFADX from the P PMA1 promoter cultivated for 72 hours in glucose and for 168 hours in crude glycerol, respectively. Furthermore, the detailed lipid analysis revealed a high enrichment of punicic acid in the triacylglycerol lipid structures, even without substantial modifications to the metabolic pathways. Conclusions This report demonstrates the high potential of R. toruloides in the biotransformation of a low-cost substrate, crude glycerol, into a value-added products such as punicic acid. The findings support the feasibility of using engineered R. toruloides as a sustainable and efficient platform for the production of punicic acid-enriched single-cell oil. Synthetic biology Metabolic engineering Rhodosporidium toruloides conjugated fatty acids (CLNA) FADX single-cell oil lipid crude glycerol Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Yeasts are commonly utilized in various biotechnological applications, including the production of biofuels, specialty chemicals, polymers, biomaterials, pharmaceuticals, enzymes, and recombinant proteins. They efficiently metabolize sugars as well as other carbon sources and, thanks to modern methods of genetic engineering, are sustainable, renewable, and have minimal environmental impact [1]. The oleaginous yeast Rhodotorula ( Rhodosporidium ) toruloides is capable of high-density growth while simultaneously producing a high titer of lipids [2]. It is also known as “the red yeast” due to its production of carotenoids, which give it its characteristic red coloration. Carotenoids, including beta-carotene, torulene, and torularhodin, are valuable for their antioxidant properties and their applications in the food, pharmaceutical, and cosmetic industries [3,4]. In addition, R. toruloides naturally utilizes various carbon sources for growth, including glucose, xylose, cellobiose, glycerol, acetic acid, and cellulose biomass hydrolysates [5–8], and exhibits halotolerance [9]. Like other yeasts, significant lipid accumulation occurs under nitrogen-limited conditions [10]. R. toruloides has immense potential for the industrial production of value-added lipids. Every year, advances in molecular tools simplify genetic manipulations with this yeast, overcoming challenges in its utilization in both scientific research and industry [11]. Punicic acid (PuA, C18:3Delta9cis,11trans,13cis) with three conjugated double bonds is an isomer of alpha-linolenic acid (C18:3Delta9cis,12cis,15cis), and it constitutes approximately 60-80% of pomegranate seed oil (PSO) [12]. Oils enriched with conjugated fatty acids are valuable for their nutritional value and industrial applications. Currently, significant attention is focused on the anticancer effects of PSO. It inhibits oxidation and prostaglandin synthesis, reduces the incidence of breast, prostate, and colon cancer, and increases the apoptosis of cancer cells [13,14]. Recent studies also suggest that PuA could be used in the prevention and treatment of neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's [15]. In pomegranate, PuA is formed by the activity of delta12-desaturase of oleic acid (FAD2) homologue, referred to as PgFADX (fatty acid conjugase) [16,17]. This enzyme converts the delta12 double bond of linoleic acid (C18:2Delta9cis,12cis) within the phosphatidylcholine molecule into two conjugated double bonds at positions C11 and C13. PgFADX exhibits dual activity; it catalyzes the production of PuA and converts oleic acid into linoleic acid, the precursor of PuA. The heterologous expression of the PgFADX in the Saccharomyces cerevisiae yeast has demonstrated that this yeast produces PuA in very limited amounts (0.8% of total fatty acids (TFA)) and only when grown in the presence of linoleic acid. Further efforts to enhance PuA production in recombinant S. cerevisiae cells (a deletion mutant in the transcription factor Snf2p simultaneously expressing PgFADX , PgPDAT , and PgLPCAT with the addition of 0.05% linoleic acid) led to a PuA accumulation reaching 3.37% of TFA [18]. Heterologous expression of the PgFADX in the Arabidopsis thaliana plant revealed that PgFADX inhibits the naturally occurring desaturase AtFAD2 [19]. An increased PuA accumulation was achieved in A. thaliana when PgFADX and PgFAD2 were simultaneously expressed in seed lines with a significantly elevated level of linoleic acid. Recently, in a modified strain of Brassica napus with a higher oleic acid content, the co-expression of PgFAD2 and PgFADX achieved 11.1% of PuA of TFA [20]. The PuA distribution in recombinant plants was high in phospholipids compared to pomegranate and low in triacylglycerols (TAG). These results suggest that lipid metabolism regulation, substrate availability, and intracellular channeling of PuA from phospholipids to TAG are essential processes for effective PuA accumulation in pomegranate seeds. Initial attempts to produce PuA in the fission yeast Schizosaccharomyces pombe showed that, unlike the heterologous expression of PgFAD2 , yeasts which express PgFADX exhibited slowed growth [21]. Recombinant S. pombe cells accumulated 38.7 mg/L of PuA, corresponding to 19.6% of PuA in TFA when PgFADX was expressed, and 34.3 mg/L of PuA, corresponding to 25.1% of PuA in TFA, in the case of the co-expression of PgFADX with PgFAD2 . The dynamics of PuA accumulation in these heterologous strains were different and correlated with the growth defects of the yeast cultures tested. This difference is likely due to an inefficient deposition of PuA into TAG, leading to PuA accumulation in phospholipids, as well as in the free fatty acid fraction. Accumulation of PuA in membrane lipids and free fatty acids can lead to PuA lipotoxicity, potentially resulting from its interference with essential cellular processes. In an effort to produce PuA by oleaginous organisms, recombinant strains of Yarrowia lipolytica were constructed [22]. The promoter optimization for PgFADX expression led to an improved PuA accumulation, from 0.9 to 1.8 mg/g of dry cell weight (DCW). The strain with the highest PuA production, expressing PgFADX under the control of a strong erythritol-inducible promoter, accumulated 36.6 mg/L of PuA. A recent study demonstrated that the yeast Y. lipolytica is a promising host for PuA production [23]. The resulting production strain with substantial multi-level genetic optimalization, such as an increased linoleic acid content, multiple integrations of PgFADX , blocked beta-oxidation, and several pathway modifications for acyl-chain editing, produced 100.6 mg/L of PuA (4.77% of TFA) in shake flask conditions, and 3072.72 mg/L of PuA in a fermenter. In this study, we constructed recombinant R. toruloides strains for PuA production without disrupting the synthesis of carotenoids, which can prevent lipid oxidation during the downstream processes of PuA isolation. The effect of three different promoters and two carbon sources were tested. In the best PuA-producing strains cultivated in media containing 6% glucose and 6% waste crude glycerol, PuA accumulation reached 105.77 mg/L (6.06 mg/g DCW, 1.32% of TFA) and 72.81 mg/L (3.56 mg/g DCW, 0.68% of TFA), respectively. The levels achieved were comparable to those recently reported for engineered Y. lipolytica strains in flask experiments, even without substantial metabolic pathway modifications. This suggests that recombinant R. toruloides is a more suitable oleaginous yeast for sustainable PuA production compared to Y. lipolytica . Methods Strains, media and cultivation Strains used in this study are listed in Table 1 . NEB 5-alpha Escherichia coli cells were used for plasmid construction. Agrobacterium tumefaciens EHA105 [24] were used for A. tumefaciens -mediated transformation (ATMT) of Rhodotorula/Rhodosporidium toruloides . R. toruloides IFO0880 (also known as APA2687 or NBRC0880) was a starting strain for all subsequent genetic modifications. E. coli and A. tumefaciens cells were cultivated at 37 °C and 30 °C in Luria Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl), respectively. To maintain the plasmids, the LB medium was supplemented with 50 mg/L kanamycin. R. toruloides was cultivated at 30 °C, 160 rpm in the YPD medium (20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract). Cell growth was estimated spectrophotometrically (Spectrophotometer, Biochrom Libra S2, Cambridge, UK) by measuring the OD at 600 nm. The yeast inoculum was prepared in 5 mL of the YPD medium in a microbial tube and 18 h old inoculum was used for the inoculation of 50 mL production media MedA + to OD of 0.5 in a 250 mL baffled Erlenmeyer flask. The production MedA + growth medium was prepared by a modification of the MedA medium [25] to obtain a high C/N ratio leading to an increased accumulation of lipids in yeasts. The lipid production MedA + medium consisted of 1.5 g/L yeast extract, 0.5 g/L NH 4 Cl, 7 g/L KH 2 PO 4 , 5 g/L Na 2 HPO 4 .12H 2 O, 0.1 g/L CaCl 2 , 1.5 g/L MgSO 4 .7H2O, 10 mg/L ZnSO 4 .7H 2 O, 0.6 mg/L FeCl 3 .6H 2 O, 0.07 mg/L MnSO 4 .H 2 O, 0.04 mg/L CuSO 4 .5H 2 O, and the carbon source, at a concentration of 60 g/L, was either glucose (Slavus, Bratislava, Slovakia) or crude glycerol (Mikrochem, Pezinok, Slovakia). For the nitrate reductase promoter, NH 4 Cl was replaced by 0.8 g/L NaNO 3 in a MedA + medium. Cells were cultivated at 30 °C and 160 rpm inside an orbital shaker (Innova 40, Hamburg, Germany) for 24 to 168 h depending on the experiment. DCW was measured gravimetrically. The residual glucose in cell-free supernatant was determined using the GlucCell glucose monitoring system (Chemglass Life Sciences, New Jersey, USA), as previously reported [26]. Table 1. Strains used in this study Strain Characteristics Source Escherichia coli NEB 5-alpha fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 NEB #C2987 Agrobacterium tumefaciens EHA105 derivative of A281 (A136/pTiBo542) Skerker J.M., UC Berkeley EHA105-PGI1-PgFADX EHA105/pZPK-P PGI1 - PgFADX -T PMA1 -P GPD1 - HYG -T NOS This study EHA105-NAR1-PgFADX EHA105/pZPK-P NAR1 - PgFADX -T PMA1 -P GPD1 - HYG -T NOS This study EHA105-PMA1-PgFADX EHA105/pZPK-P PMA1 - PgFADX -T PMA1 -P GPD1 - HYG -T NOS This study Rhodotorula toruloides IFO0880 MAT A2 Skerker J.M., UC Berkeley PGI4–PGI36 IFO0880/P PGI1 - PgFADX -T PMA1 -P GPD1 - HYG -T NOS cassette This study NAR10–NAR38 IFO0880/P NAR1 - PgFADX -T PMA1 -P GPD1 - HYG -T NOS cassette This study PMA1­­­­­–PMA12 IFO0880/P PMA1 - PgFADX -T PMA1 -P GPD1 - HYG -T NOS cassette This study Plasmid construction, transformation, and verification The PgFADX was codon-optimized according to the codon preference of R. toruloides and synthetized by Generay Biotech (Shanghai, China). The terminator sequence of the plasma membrane H + -ATPase (T PMA1 ), the promoter sequences of glucose-6-phosphate isomerase (P PGI1 ), the nitrate reductase (P NAR1 ), and the plasma membrane H + -ATPase (P PMA1 ) were amplified from the genomic DNA of R. toruloides using the primer pairs listed in Supplementary Table S1 . The codon optimized PgFADX sequence, the promoter and the terminator sequences are listed in Supplementary Table S2 . To construct the plasmids pZPK-P PGI1 - PgFADX -T PMA1 -P GPD1 - HYG -T NOS , pZPK-P NAR1 - PgFADX -T PMA1 -P GPD1 - HYG -T NOS , and pZPK-P PMA1 - PgFADX -T PMA1 -P GPD1 - HYG -T NOS, three fragments consisting of the corresponding promoter sequence, PgFADX , and T PMA1 were assembled into the binary linearized vector pZPK-P GPD1 - HYG -T NOS using the Gibson assembly reaction (E5520S, New England Biolabs, MA, USA). The plasmids were amplified in E. coli , electroporated into A. tumefaciens , and then used to transform R. toruloides IFO0880 according to the ATMT, as described previously [27]. The transformants were resuspended in YPD and spread on YPD plates supplemented with 50 μg/mL hygromycin, 300 μg/mL cefotaxime, and 300 μg/mL carbenicillin to recover single clones. To verify the integration of the expected DNA fragment, R. toruloides colonies were subjected to colony-PCR with the primers listed in Supplementary Table S1 , according to the previously described method [28]. Lipid extraction procedure Lipids were extracted using a method previously reported, with minor modifications [21]. Briefly, yeast cells (aliquots of OD 50 - corresponding to approximately 10-13 mg of DCW) were collected by centrifugation, washed, and the cell pellets were frozen. The cells were suspended in 1 mL of a mixture of chloroform and methanol (2:1, v/v) containing the antioxidant butylated hydroxytoluene (BHT) at a final concentration of 0.01% and disrupted by FastPrep disintegrator (MP Biomedicals) with glass beads (diameter 0.4 mm, 3x40 s at the highest speed, with 5 min cooling on ice between cycles) to obtain homogenates. For the thin layer chromatography (TLC), lipids were extracted from homogenates by chloroform/methanol/water (1:2:0.8, v/v) and subsequently the proportion of the mixture was adjusted to 2:2:1.8 (v/v) at room temperature, according to the procedure of [29]. The organic phase containing the lipids was separated by centrifugation; the lipids were dried under a stream of N 2, and the dry lipids were dissolved in a 100 μL mixture of chloroform and methanol (2:1, v/v) and BHT, prior to use. Analytical methods For TLC analyses, an aliquot of lipid extract corresponding to 0.5 mg of DCW for neutral lipids and 3 mg of DCW for phospholipids was applied to Silica Gel 60 TLC plates (Merck, Darmstadt, Germany) using a Linomat 5 semiautomatic sample applicator (CAMAG Linomat 5, Muttenz, Switzerland). Neutral lipids were separated by a two-step TLC solvent system using a method described previously (first step: petroleum ether/diethyl ether/acetic acid, 70:30:2; second step: petroleum ether and diethyl ether, 49:1) [30]. Individual lipid spots were visualized by charring the plates, as previously reported [31]. Phospholipids were separated by the solvent system (chloroform/methanol/acetic acid/water, 75:45:3:1), as described previously [32]. Individual lipid spots were identified using lipid standards. The presence of PuA in individual lipids was determined by densitometry scan at 276 nm (CAMAG TLC Scanner 3, Muttenz, Switzerland). For fatty acid analysis, the total lipid homogenate, corresponding to approximately 10 mg DCW, was transmethylated with 5% Na-OCH 3 in methanol. Fatty acid methyl esters (FAME) were then extracted using n -hexane, as described previously [17]. The analysis of FAME was performed by the injection of 1 μL aliquots into a gas chromatography (GC) apparatus (GC2010Plus, Shimadzu) equipped with a BPX70 capillary column (30 m × 0.25 mm × 0.25 µm, SGE Analytical Science), as described previously [21,33]. Individual FAMEs were identified by comparing them with authentic standards (C4−C24 FAME mixture, Supelco). The quantification of individual fatty acids was conducted using tridecanoic acid methyl ester as an internal standard (Merck, Darmstadt, Germany). To determine the relative fatty acids content in TAG and phospholipids, the corresponding lipid spots were scraped off the TLC plate into glass tubes and transmethylated, as described above. Fluorescence microscopy The R. toruloides w ild type IFO0880 strain was cultured in a YPD media or in a MedA + media containing 6% glucose or glycerol for 48 h. The cell culture was diluted to OD = 2.0, harvested by centrifugation, washed once with a 50 mM Tris-HCl, pH 7.5, and suspended in 0.3 mL of 50 mM Tris-HCl, pH 7.5. LD540 (a stock solution of 0.05 mg/mL in ethanol) was added to the final concentration of 1 µg/mL, and the cells were incubated in the dark for 15 min at room temperature. A 3 µL drop of cell suspension was examined for the presence of the lipid droplets using a Leica DM5500 fluorescence microscope equipped with an HCX PL Fluotar 100× objective, and a Leica DFC340 FX digital camera. Signals were detected using the filter system Y3 for CY3 green. All images were captured at the identical instrument settings and processed using LAS 3.0 software (Leica Microsystems, Wetzlar, Germany). Statistics and reproducibility All experiments were performed at least in duplicates. The data was expressed as means ± standard errors. All data analysis was performed by Excel. Results And Discussion Promoters selection and strains construction The strain IFO0880 natively accumulates lipids at higher titers [8] and was therefore utilized for the engineering of PuA production. First, three types of plasmids based on the pZPK-P GPD1 - HYG -T NOS backbone were constructed, with PgFADX expression controlled by three different promoters. The choice of promoter is directly related to the expression level of the target protein [34,35]. An appropriately selected promoter can result in higher yields of the desired product without adversely affecting cell growth. So far, several promoters, including constitutive and inducible types, have been characterized in R. toruloides . One of the promoters selected was a constitutive P PGI1 promoter of the glucose 6-phosphate isomerase. This promoter was previously shown to be four times stronger than the promoter of the glyceraldehyde 3-phosphate dehydrogenase, P GPD1 , when driving the expression of HYG in cells supplemented with an increased concentration of hygromycin [36]. Since, in the effort to produce conjugated linolenic acid isomers (CLNA) in recombinant fission yeast, it was shown that a high accumulation of PuA and calendic acid influences cell growth [21,37], the second promoter selected was an inducible P NAR1 of the nitrate reductase regulated by the nitrogen source [38]. The third promoter used was P PMA1 of the plasma membrane proton-transporting ATPase, which is often employed as a constitutive promoter in the model yeast S. cerevisiae . The expression cassettes containing codon optimized PgFADX under the control of the three different selected promoters were randomly integrated into the R. toruloides IFO0880 genome using the ATMT. After selection of the stable transformants, the presence of an insertion cassette in the genomic DNA was confirmed by PCR analysis. Screening of the transformants expressing PgFADX Initially, we analyzed whether R. toruloides accumulates lipid storage organelles, lipid droplets, in a nitrogen-limited MedA + medium, which is used to stimulate the formation of lipids in oleaginous yeast Y. lipolytica [39]. Lipid droplets were observed in living cells by staining with a lipid droplet-specific dye LD540. As shown in Fig. 1 , the R. toruloides IFO0880 cultured in a MedA + medium showed enlarged lipid droplets compared to cells grown in a rich YPD medium. The presence of lipid droplets of bigger sizes was detected in cells grown in MedA + containing crude glycerol as a carbon source. Taken together, our results suggest that a MedA + medium supplemented with glucose or crude glycerol could be a suitable medium for the heterologous production of a single-cell oil containing PuA using metabolic engineering techniques. Secondly, twelve randomly selected PgFADX -containing transformants for each promoter were screened for their ability to produce PuA. This was because transformants obtained by the ATMT usually have the expression cassette randomly integrated into the genome, which can influence cell growth and the expression of the desired gene from the cassette [40]. As shown in Fig. 2 , randomly picked PgFADX -containing transformants accumulated comparable levels of TFA per cell growth as wild type IFO0880. The most important result was the successful production of PuA in all tested engineered recombinant strains. Generally, a relatively higher content of PuA in TFA was observed for transformants expressing PgFADX under the control of the P PMA1 promoter. The use of the P PMA1 promoter enhanced the production of PuA and the best PuA-producing strain, PMA5, accumulated 8.6-fold and 11.1-fold more µg/OD PuA compared to the PuA best-producing strains of the other two promoters, PGI28, and NAR13, respectively. This result confirmed the importance of promoter choice and suitable screening of engineered strains when ATMT transformation is used to obtain transformants with random integration of the desired cassette. Accumulation and distribution of PuA in production media containing glucose It was previously shown that the production of CLNA is a dynamic process [21,23,37]. Therefore, PuA-producing transformants were analyzed for the dynamics of PuA production in a time dependent manner. Two transformants for each promoter were selected, namely PGI26 and PGI28, NAR13 and NAR16, and PMA5 and PMA6, which express PgFADX from P PGI , P NAR1 , and P PMA1 promoters, respectively. First, the growth, biomass yield, glucose consumption, and fatty acid production in the engineered strains were compared to the wild type strain IFO0880 ( Fig. 3 and Table 2 ). All the selected recombinant strains containing randomly integrated PgFADX- expression cassettes grew comparably well, with a slight increase in OD and biomass compared to the wild type strain. Most of the glucose was consumed after 72 h cultivation. All strains accumulated, on average, 40-50% of TFA in biomass. The fatty acid profile of the wild type strain remained stable during the cultivation periods of 72 h, 120 h, and 168 h ( Supplementary Table S3 ). A substantial difference was observed in the ratio of monounsaturated to polyunsaturated fatty acid (MUFA/PUFA) for the PuA-producing recombinant strains. The expression of PgFADX resulted in an increased relative content of oleic acid (C18:1) and decreased levels of linoleic acid (C18:2) and α-linolenic acid (C18:3). These observed changes correlated with the elevation of PuA levels in the recombinant cells. This result suggests that the PgFADX might compete for the C18:1 substrate with the activity of the endogenous FAD2 in the engineered strains, similar to what observed in recombinant Arabidopsis thaliana plants [19]. The highest relative content of PuA reached 1.3% at 72 h and 120 h in the PMA6 strain containing the P PMA1 promoter, which was much higher than in the strains with PgFADX expressed from the P PGI and P NAR1 promoters ( Table 2 ). When productivity is taken into consideration, in the best PuA-producing strain PMA6, the total PuA reached 105.8 mg/L and 6.1 mg/g DCW at 30°C after 72 h ( Table 2 ). Similar results were obtained with prolonged cultivation times of 120 h and 168 h, suggesting that the PuA level is stable over time in R. toruloides cells. The expression under P PGI and P NAR1 promoters showed a 10-fold and 9-fold lower production of PuA, respectively, compared to the P PMA1 promoter. Table 2. Fatty acid accumulation in strains cultivated in MedA + medium containing 6% glucose. Strain PgFADX promoter Time TFA (g/L) TFA/DCW (%) PuA (% of TFA) PuA (mg/g DCW) PuA (mg/L) IFO0880 - 72 h 4.55 ± 0.17 38.81 ± 0.97 - - - 120 h 6.20 ± 0.02 41.80 ± 1.86 - - - 168 h 6.61 ± 0.38 42.22 ± 2.20 - - - PGI26 P PGI1 72 h 7.54 ± 0.08 46.09 ± 3.85 0.13 ± 0.00 0.59 ± 0.03 9.74 ± 0.49 120 h 8.44 ± 0.62 45.23 ± 1.67 0.12 ± 0.00 0.55 ± 0.02 10.35 ± 0.71 168 h 7.26 ± 1.36 40.46 ± 9.80 0.12 ± 0.00 0.49 ± 0.12 8.70 ± 1.74 PGI28 P PGI1 72 h 6.76 ± 0.67 37.97 ± 1.89 0.10 ± 0.00 0.37 ± 0.00 6.65 ± 0.40 120 h 8.57 ± 0.71 45.05 ± 2.36 0.10 ± 0.00 0.43 ± 0.02 8.21 ± 0.66 168 h 7.43 ± 1.02 39.71 ± 5.44 0.09 ± 0.00 0.37 ± 0.05 6.90 ± 0.90 NAR13 P NAR1 72 h 9.10 ± 0.55 48.50 ± 2.58 0.13 ± 0.00 0.62 ± 0.01 11.62 ± 0.09 120 h 8.69 ± 0.27 46.81 ± 1.95 0.13 ± 0.01 0.61 ± 0.03 11.42 ± 0.61 168 h 8.75 ± 0.55 44.84 ± 2.54 0.13 ± 0.00 0.59 ± 0.05 11.42 ± 1.03 NAR16 P NAR1 72 h 9.27 ± 0.22 46.53 ± 0.24 0.07 ± 0.00 0.33 ± 0.00 6.59 ± 0.09 120 h 9.53 ± 0.10 49.86 ± 2.45 0.07 ± 0.00 0.36 ± 0.03 6.97 ± 0.29 168 h 8.29 ± 0.65 43.16 ± 2.44 0.07 ± 0.00 0.31 ± 0.03 6.04 ± 0.61 PMA5 P PMA1 72 h 7.35 ± 0.51 46.16 ± 3.71 0.97 ± 0.11 4.45 ± 0.13 70.91 ± 2.86 120 h 7.39 ± 0.53 42.09 ± 5.66 0.98 ± 0.01 4.14 ± 0.62 72.70 ± 6.34 168 h 6.46 ± 2.10 35.26 ± 6.29 0.96 ± 0.01 3.37 ± 0.58 61.63 ± 19.63 PMA6 P PMA1 72 h 8.02 ± 0.58 45.98 ± 2.65 1.32 ± 0.06 6.06 ± 0.07 105.77 ± 2.81 120 h 8.17 ± 0.17 44.05 ± 1.41 1.27 ± 0.03 5.60 ± 0.30 103.90 ± 4.48 168 h 7.63 ± 0.74 42.39 ± 5.67 1.25 ± 0.02 5.28 ± 0.80 95.00 ± 10.78 Abbreviations: DCW, dry cell weight; PuA, punicic acid; TFA, total fatty acids. It is worth emphasizing that the PuA yield was 5.4-fold higher than the yield obtained in recombinant S. pombe over-expressing PgFADX from the strong inducible NMT1 promoter [21], and 2.9-fold higher than in the recombinant obese Y. lipolytica strain expressing PgFADX from the hybrid inducible pEYK1 4AB-coreTEF promoter [22]. It is proposed that the main limitation in PuA accumulation is the inefficient flux of PuA from phospholipids to TAG in recombinant yeasts. This hypothesis was proven in the recombinant model yeast S. cerevisiae [18] and more recently in the recombinant oleaginous yeast Y. lipolytica , which can accumulate increased levels of PuA only after significant multi-level genetic optimalization, including an improved supply of C18:2, expressing multiple copies of PgFADX , the acyl-editing, β-oxidation, and glycerol-3-phosphate synthesis pathways [23]. To sum up, our results demonstrated that the recombinant oleaginous red yeast R. toruloides, expressing PgFADX from a P PMA1 promoter, is capable of producing PuA yields comparable to those recently reported for the engineered oleaginous yeast Y. lipolytica, with comprehensive genetic refinement. The relative level of PuA in engineered R. toruloides is not high; therefore, it is expected that further optimization of the metabolic pathways, including but not limited to the carbon and C18:2 supply, enhanced PuA synthesis, and PuA channeling to TAG lipid structures, could lead to a significant increase in PuA titer. CLNA are preferentially synthesized through biotransformation of linoleic acid esterified to phosphatidylcholine in native producers and then very efficiently channeled from membrane phospholipids to lipid storage depots, lipid droplets, in the form of TAG [19,41]. However, the precise mechanism of CLNA channeling is still not well understood and is currently under investigation. Previous studies have demonstrated a significant difference in the relative content of PuA in TAG and phosphatidylcholine lipid structures between pomegranate seeds, which naturally produce PuA, and the seeds of transgenic plants [19]. Therefore, the relative content of PuA in TAG and phospholipids was examined. In order to characterize the distribution of PuA in individual lipid classes, the presence of conjugated double bonds in the PuA structure, which allows its detection under UV light, was utilized [42]. First, total lipid extracts of R. toruloides were separated on TLC plates to analyze the phospholipids. With this approach, PuA was mainly detected in the phosphatidylcholine ( Fig. 4A ), which is reported as the primary place for CLNA synthesis. Second, the total lipid extracts were loaded on a TLC plate and the plate was developed in conditions favoring the separation of neutral lipids. The majority of PuA was detected in TAG ( Fig. 4B ). The presence of PuA in steryl esters could not be verified due to possible interference from the signal of some sterol molecules containing conjugated double bonds. Furthermore, PuA in a pool of free fatty acids was negligible. This is in contrast with the presence of CLNA in lipid extracts from recombinant yeast strains accumulating substantial amounts of free CLNA [22,37,43]. It is worth noting that, although the relative amount of PuA in engineered R. toruloides strains is not high, the UV signal indicates that PuA is predominantly distributed in TAG lipid structures ( Fig. 4A and B ). This result suggests that, in engineered R. toruloides strains, the PuA is efficiently channeled from the site of synthesis (phosphatidylcholine) to the TAG lipid structures. However, this might be due to the very high ratio of TAG to phospholipids in engineered strains under tested conditions. To further analyze the distribution of PuA in lipid fractions in the two best PuA-producing strains, PMA5 and PMA6, the relative fatty acid content in phospholipids and TAG was determined ( Fig. 4C and 4D ). In both strains, the relative content of C18:1 increased in the lipid fractions examined compared to the wild-type strain, while the relative content of C18:3 showed an almost 4-fold decrease. The PuA contents in both strains were below 0.1% and approximately 1% in the phospholipids and TAG fractions, respectively. The high enrichment of PuA in the TAG lipid structures and, at the same time, the high yield of PuA with a minimal requirement for genome modification suggests that the red yeast R. toruloides is a more suitable oleaginous yeast for PuA production than the yeast Y. lipolytica . Accumulation and distribution of PuA in production media containing crude glycerol A byproduct of biodiesel production, a crude glycerol can be utilized for microbial lipid production using the yeast R. toruloides [5,44]. Converting a low-cost carbon source into a value-added product, such as PuA, could significantly reduce the upstream expenses, thereby making the overall production process more economically viable. In our initial experiments, the accumulation of enlarged lipid droplets was observed in the R. toruloides wild type IFO0880 strain, grown in a MedA + medium supplemented with 6% crude glycerol ( Fig. 1 ). Since the response of promoters to different carbon source may impact the final yield of the desired product [34,35] the PuA production efficiency, with the glucose replaced with crude glycerol, was evaluated. The recombinant strains producing the highest PuA yields for all three promoters were examined for growth, TFA accumulation, PuA content and its distribution. The procedure was similar to that used with the MedA + medium supplemented with glucose. As shown on Fig. 5A , the growth of PuA-producing R. toruloides strains was comparable to the growth of the wild type IFO0880. Compared to the glucose-containing medium ( Fig. 3 ), the growth in crude glycerol was slightly slower, but after 120 h, the cells reached a similar OD as when grown in glucose. The biomass yield and lipid accumulation increased with cultivation time ( Fig. 5B ) and strains accumulated, on average, approximately 40-50% of TFA in biomass at the 120 h and 168 h time points ( Table 3 ) . This result confirms that crude glycerol is a suitable low-cost carbon source for microbial lipid production using R. toruloides . Similar to the glucose medium, the relative content of fatty acids in the wild type strain remained stable during the cultivation periods of 72 h, 120 h, and 168 h ( Supplementary Table S4 ). However, the relative content of PUFA decreased in the MedA + supplemented with glycerol compared to the glucose medium. In general, the amount of PuA increased with the cultivation time ( Table 3 ). In comparison to the glucose medium, the PuA yield significantly increased in strains with PgFADX expressed from the P NAR1 promoter when cultivated on a low-cost crude glycerol medium. The highest relative level of PuA was 0.9% of TFA at 72 h in the PMA5 strain containing the P PMA1 promoter, which was again much higher than in the strains with PgFADX expressed from the P PGI and P NAR1 promoters. When productivity is taken into consideration, the best PuA-producing strain, PMA5, achieved a total PuA yield of 72.8 mg/L and 3.6 mg/g DCW at 30°C after 168 h in shake flask conditions. The level of PuA obtained is approximately two-fold higher than that has been reported for the engineered S. pombe [21], and Y. lipolytica [22] strains. However, it is about one-third lower than the yields reported in a recent study on recombinant Y. lipolytica , which achieved higher yields through significant optimization of the metabolic pathways grown with glucose as a carbon source in shake flask conditions [23]. Nevertheless, it should be noted that, considering the upstream costs for the carbon source, the yield obtained from low-cost crude glycerol is more economically viable, despite the lower PuA levels achieved. Table 3. Fatty acid accumulation in strains cultivated in MedA + medium containing 6% crude glycerol. Strain PgFADX promoter Time TFA (g/L) TFA/DCW (%) PuA (% of TFA) PuA (mg/g DCW) PuA (mg/L) IFO0880 - 72 h 5.26 ± 0.15 39.40 ± 0.12 - - - 120 h 9.19 ± 0.21 48.73 ± 0.79 - - - 168 h 11.71 ± 1.12 52.57 ± 4.33 - - - PGI26 P PGI1 72 h 4.92 ± 0.08 39.11 ± 0.35 0.04 ± 0.00 0.17 ± 0.00 2.10 ± 0.01 120 h 8.43 ± 0.97 46.38 ± 0.50 0.03 ± 0.00 0.15 ± 0.00 2.76 ± 0.29 168 h 8.84 ± 1.32 49.67 ± 2.83 0.03 ± 0.00 0.16 ± 0.01 2.93 ± 0.38 PGI28 P PGI1 72 h 4.85 ± 0.35 38.53 ± 0.06 0.04 ± 0.01 0.14 ± 0.01 1.73 ± 0.01 120 h 8.31 ± 0.74 43.18 ± 1.89 0.03 ± 0.01 0.11 ± 0.01 2.14 ± 0.09 168 h 9.82 ± 2.71 49.70 ± 4.23 0.03 ± 0.01 0.12 ± 0.01 2.44 ± 0.57 NAR13 P NAR1 72 h 3.00 ± 0.03 31.65 ± 1.50 0.14 ± 0.01 0.43 ± 0.02 4.05 ± 0.03 120 h 6.98 ± 1.12 41.43 ± 2.18 0.14 ± 0.01 0.58 ± 0.08 9.70 ± 0.67 168 h 8.20 ± 1.99 50.20 ± 4.98 0.26 ± 0.04 1.26 ± 0.03 20.44 ± 2.48 NAR16 P NAR1 72 h 3.16 ± 0.03 33.71 ± 0.90 0.09 ± 0.01 0.29 ± 0.04 2.68 ± 0.25 120 h 7.88 ± 1.68 42.08 ± 0.67 0.13 ± 0.01 0.53 ± 0.01 9.91 ± 1.87 168 h 8.79 ± 1.60 48.90 ± 1.10 0.29 ± 0.02 1.40 ± 0.07 24.98 ± 2.69 PMA5 P PMA1 72 h 5.22 ± 0.25 37.33 ± 1.14 0.89 ± 0.08 3.33 ± 0.21 46.54 ± 2.13 120 h 9.06 ± 0.97 48.95 ± 1.56 0.69 ± 0.06 3.35 ± 0.19 61.74 ± 1.18 168 h 10.86 ± 1.96 52.83 ± 1.64 0.68 ± 0.06 3.56 ± 0.19 72.81 ± 7.12 PMA6 P PMA1 72 h 5.17 ± 0.11 34.88 ± 0.12 0.75 ± 0.00 2.63 ± 0.01 38.94 ± 0.86 120 h 8.46 ± 0.13 41.64 ± 2.68 0.60 ± 0.00 2.48 ± 0.16 50.43 ± 0.75 168 h 10.27 ± 0.89 47.96 ± 2.99 0.61 ± 0.01 2.92 ± 0.10 62.44 ± 3.56 Abbreviations: DCW, dry cell weight; PuA, punicic acid; TFA, total fatty acids. To examine the distribution of PuA in individual lipid classes, the total lipids were separated on TLC plates and the detection of PuA under UV light was used, as described above. Similarly, as with the glucose-containing medium, the signal for PuA was mainly detected in phosphatidylcholine ( Fig. 6A ) and TAG lipid structures ( Fig. 6B ). The signal for free PuA was negligible. Next, the relative contents of PuA in phospholipid and TAG fractions in the two best PuA-producing strains, PMA5 and PMA6, was analyzed ( Fig. 6C and 6D ). The PuA content in both strains was approximately 0.2% and 0.6% in the phospholipids and TAG fractions, respectively. The enrichment of PuA in the single-cell oil, which are comprised of TAG lipid structures, was not as high as in the glucose medium. However, since the TAG are predominant lipid structures in R. toruloides, grown under nitrogen-limited conditions, with glycerol as the carbon source, the majority of PuA is stored in TAG lipid structures. Our results confirmed that the medium with a low-cost carbon source is suitable for the high production of PuA by the recombinant red yeast R. toruloides . The price of media components, mainly carbon and nitrogen sources, is a critical factor in any biotechnological process [44,45]. R. toruloides exhibits greater versatility than Y. lipolytica in assimilating low-cost carbon substrates, including crude glycerol, lignocellulosic hydrolysates, and molasses [2]. This versatility significantly enhances its potential use in various biotechnological applications. Moreover, R. toruloides can accumulate more lipids from crude glycerol than from pure glycerol, without being negatively affected by the impurities present in crude glycerol [46]. An additional advantage of R. toruloides is its natural production of carotenoids, which act as antioxidants and are widely used in food, pharmaceuticals, and cosmetics [3,4]. It has been demonstrated that carotenoids can effectively inhibit lipid peroxidation [47]. Therefore, carotenoids, which naturally co-purify with lipids during the lipid extraction from engineered R. toruloides cells (data not shown), could prevent PuA oxidation of PuA-enriched single cell oil. Another notable difference between the two oleaginous yeasts is that R. toruloides naturally synthesizes C18:3, unlike Y. lipolytica . It is well documented that the level of unsaturation in PUFA correlates with increased membrane fluidity, elasticity, and flexibility [48]. Therefore, C18:3 has a greater impact on membrane properties compared to C18:2. It can be hypothesized that, due to this difference, R. toruloides efficiently channels C18:3 from phospholipids to TAG lipid structures to maintain membrane properties. The presence of conjugated double bonds in PuA further impacts the cellular membrane properties. Considering these factors, along with the high accumulation of PuA in engineered R. toruloides cells, it is evident that R. toruloides may have even greater biotechnological potential for PuA production than Y. lipolytica . Conclusion For the first time, to our knowledge, the low-cost substrate, crude glycerol, was used for the production of a single-cell oil enriched in PuA using the recombinant red yeast R. toruloides . The use of a promoter for plasma membrane ATPase for the expression of PgFADX improved the overall productivity of the recombinant strain. The high PuA yield and its enrichment in TAG lipid structures, together with the minimal requirements necessary for genome modification, suggests that the red yeast R. toruloides is a more suitable host for PuA production compared to Y. lipolytica . In addition, the red yeast naturally synthesizes carotenoids, which possess nutritional value, and can prevent oxidation of the CLNA during the downstream processing of lipids. With the further optimalization of the metabolic pathways, and medium formulation, further improvements of red yeast R. toruloides for sustainable PuA production are expected. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Data availability The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Acknowledgements The authors thank to Gina Geiselman (Sandia National Laboratories, Livermore, USA) for providing a detailed ATMT transformation protocol of R. toruloides , Milan Certik and Peter Gajdos (Slovak Technical University, Bratislava, Slovakia) for providing the codon optimized PgFADX sequence, Jeffrey Michael Skerker (University of California, Berkeley, US), and Zongbao Kent Zhao (Dalian University of Technology, Dalian, China) for kindly providing strains and cloning vector used in this study. Funding This work was financially supported by the Slovak Research and Development Agency under the contract No. APVV-20-0166 and the Grant Programme for SAS PhD students (APP0521). Contribution DK:Methodology, Investigation, Writing – review, Funding acquisition. RH:Visualization, Supervision, Writing – original draft, review & editing, Funding acquisition. All authors reviewed the manuscript. References Villena GK, Ludeña Y, Samolski I. Applications of yeast for environmental clean-up and sustainable agriculture. Advances in Yeast Biotechnology for Biofuels and Sustainability [Internet]. Elsevier; 2023 [cited 2024 Jun 26]. p. 193–218. Available from: https://linkinghub.elsevier.com/retrieve/pii/B9780323954495000187 Park Y-K, Nicaud J-M, Ledesma-Amaro R. The Engineering Potential of Rhodosporidium toruloides as a Workhorse for Biotechnological Applications. Trends in Biotechnology. 2018;36:304–17. Nabi F, Arain MA, Rajput N, Alagawany M, Soomro J, Umer M, et al. Health benefits of carotenoids and potential application in poultry industry: A review. Animal Physiology Nutrition. 2020;104:1809–18. Zheng X, Hu R, Chen D, Chen J, He W, Huang L, et al. Lipid and carotenoid production by the Rhodosporidium toruloides mutant in cane molasses. Bioresource Technology. 2021;326:124816. Bommareddy RR, Sabra W, Maheshwari G, Zeng A-P. Metabolic network analysis and experimental study of lipid production in Rhodosporidium toruloides grown on single and mixed substrates. Microb Cell Fact. 2015;14:36. Huang X-F, Liu J-N, Lu L-J, Peng K-M, Yang G-X, Liu J. Culture strategies for lipid production using acetic acid as sole carbon source by Rhodosporidium toruloides. Bioresource Technology. 2016;206:141–9. Xu J, Zhao X, Wang W, Du W, Liu D. Microbial conversion of biodiesel byproduct glycerol to triacylglycerols by oleaginous yeast Rhodosporidium toruloides and the individual effect of some impurities on lipid production. Biochemical Engineering Journal. 2012;65:30–6. Zhang S, Skerker JM, Rutter CD, Maurer MJ, Arkin AP, Rao CV. Engineering Rhodosporidium toruloides for increased lipid production. Biotech & Bioengineering. 2016;113:1056–66. Tchakouteu SS, Kopsahelis N, Chatzifragkou A, Kalantzi O, Stoforos NG, Koutinas AA, et al. Rhodosporidium toruloides cultivated in NaCl‐enriched glucose‐based media: Adaptation dynamics and lipid production. Engineering in Life Sciences. 2017;17:237–48. Ratledge C, Wynn JP. The Biochemistry and Molecular Biology of Lipid Accumulation in Oleaginous Microorganisms. Advances in Applied Microbiology [Internet]. Elsevier; 2002 [cited 2024 Jun 28]. p. 1–52. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0065216402510005 Wen Z, Zhang S, Odoh CK, Jin M, Zhao ZK. Rhodosporidium toruloides - A potential red yeast chassis for lipids and beyond. FEMS Yeast Research. 2020;20:foaa038. Takagi T, Itabashi Y. Occurrence of mixtures of geometrical isomers of conjugated octadecatrienoic acids in some seed oils: Analysis by open-tubular gas liquid chromatography and high performance liquid chromatography. Lipids. 1981;16:546–51. Aruna P, Venkataramanamma D, Singh AK, Singh RP. Health Benefits of Punicic Acid: A Review. Comprehensive Reviews in Food Science and Food Safety. 2016;15:16–27. Shabbir MA, Khan MR, Saeed M, Pasha I, Khalil AA, Siraj N. Punicic acid: A striking health substance to combat metabolic syndromes in humans. Lipids Health Dis. 2017;16:99. Guerra-Vazquez CM, Martinez-Avila M, Guajardo-Flores D, Antunes-Ricardo M. Punicic Acid and Its Role in the Prevention of Neurological Disorders: A Review. Foods. 2022;11. Hornung E, Pernstich C, Feussner I. Formation of conjugated Delta11Delta13-double bonds by Delta12-linoleic acid (1,4)-acyl-lipid-desaturase in pomegranate seeds. Eur J Biochem. 2002;269:4852–9. Iwabuchi M, Kohno-Murase J, Imamura J. Delta 12-oleate desaturase-related enzymes associated with formation of conjugated trans-delta 11, cis-delta 13 double bonds. J Biol Chem. 2003;278:4603–10. Wang J, Xu Y, Holic R, Yu X, Singer SD, Chen G. Improving the Production of Punicic Acid in Baker’s Yeast by Engineering Genes in Acyl Channeling Processes and Adjusting Precursor Supply. J Agric Food Chem. 2021;69:9616–24. Mietkiewska E, Miles R, Wickramarathna A, Sahibollah AF, Greer MS, Chen G, et al. Combined transgenic expression of Punica granatum conjugase (FADX) and FAD2 desaturase in high linoleic acid Arabidopsis thaliana mutant leads to increased accumulation of punicic acid. Planta. 2014;240:575–83. Xu Y, Mietkiewska E, Shah S, Weselake RJ, Chen G. Punicic acid production in Brassica napus. Metabolic Engineering. 2020;62:20–9. Garaiova M, Mietkiewska E, Weselake RJ, Holic R. Metabolic engineering of Schizosaccharomyces pombe to produce punicic acid, a conjugated fatty acid with nutraceutic properties. Appl Microbiol Biotechnol. 2017; Urbanikova V, Park Y-K, Krajciova D, Tachekort M, Certik M, Grigoras I, et al. Yarrowia lipolytica as a Platform for Punicic Acid Production. IJMS. 2023;24:8823. Wang K, Zhou Y, Cao L, Lin L, Ledesma-Amaro R, Ji X-J. Engineering Yarrowia lipolytica for Sustainable Production of the Pomegranate Seed Oil-Derived Punicic Acid. J Agric Food Chem. 2024;72:3088–98. Hood EE, Gelvin SB, Melchers LS, Hoekema A. NewAgrobacterium helper plasmids for gene transfer to plants. Transgenic Research. 1993;2:208–18. Holdsworth JE, Veenhuis M, Ratledge C. Enzyme Activities in Oleaginous Yeasts Accumulating and Utilizing Exogenous or Endogenous Lipids. Microbiology. 1988;134:2907–15. Roop JI, Chang KC, Brem RB. Polygenic evolution of a sugar specialization trade-off in yeast. Nature. 2016;530:336–9. Coradetti ST, Pinel D, Geiselman GM, Ito M, Mondo SJ, Reilly MC, et al. Functional genomics of lipid metabolism in the oleaginous yeast Rhodosporidium toruloides. eLife. 2018;7:e32110. Lin X, Wang Y, Zhang S, Zhu Z, Zhou YJ, Yang F, et al. Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides . FEMS Yeast Res. 2014;14:547–55. Bligh EG, Dyer WJ. A RAPID METHOD OF TOTAL LIPID EXTRACTION AND PURIFICATION. Can J Biochem Physiol. 1959;37:911–7. Spanova M, Czabany T, Zellnig G, Leitner E, Hapala I, Daum G. Effect of lipid particle biogenesis on the subcellular distribution of squalene in the yeast Saccharomyces cerevisiae. J Biol Chem. 2010;285:6127–33. Garaiova M, Zambojova V, Simova Z, Griac P, Hapala I. Squalene epoxidase as a target for manipulation of squalene levels in the yeast Saccharomyces cerevisiae. FEMS Yeast Res. 2014;14:310–23. Garner K, Hunt AN, Koster G, Somerharju P, Groves E, Li M, et al. Phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) binds and transfers phosphatidic acid. J Biol Chem. 2012;287:32263–76. Mietkiewska E, Siloto RM, Dewald J, Shah S, Brindley DN, Weselake RJ. Lipins from plants are phosphatidate phosphatases that restore lipid synthesis in a pah1Delta mutant strain of Saccharomyces cerevisiae. FEBS J. 2011;278:764–75. Ho P-W, Klein M, Futschik M, Nevoigt E. Glycerol positive promoters for tailored metabolic engineering of the yeast Saccharomyces cerevisiae. FEMS Yeast Research [Internet]. 2018 [cited 2024 Jun 27];18. Available from: https://academic.oup.com/femsyr/article/doi/10.1093/femsyr/foy019/4898018 Vogl T, Kickenweiz T, Pitzer J, Sturmberger L, Weninger A, Biggs BW, et al. Engineered bidirectional promoters enable rapid multi-gene co-expression optimization. Nat Commun. 2018;9:3589. Wang Y, Lin X, Zhang S, Sun W, Ma S, Zhao ZK. Cloning and evaluation of different constitutive promoters in the oleaginous yeast Rhodosporidium toruloides . Yeast. 2016;33:99–106. Garaiova M, Hua Q, Holic R. Heterologous Production of Calendic Acid Naturally Found in Calendula officinalis by Recombinant Fission Yeast. J Agric Food Chem. 2023;71:3842–51. Johns AMB, Love J, Aves SJ. Four Inducible Promoters for Controlled Gene Expression in the Oleaginous Yeast Rhodotorula toruloides. Front Microbiol. 2016;7:1666. Hambalko J, Gajdoš P, Nicaud J-M, Ledesma-Amaro R, Tupec M, Pichová I, et al. Production of Long Chain Fatty Alcohols Found in Bumblebee Pheromones by Yarrowia lipolytica. Front Bioeng Biotechnol. 2021;8:593419. Lin X, Gao N, Liu S, Zhang S, Song S, Ji C, et al. Characterization the carotenoid productions and profiles of three Rhodosporidium toruloides mutants from Agrobacterium tumefaciens-mediated transformation. Yeast. 2017;34:335–42. Yurchenko O, Shockey JM, Gidda SK, Silver MI, Chapman KD, Mullen RT, et al. Engineering the production of conjugated fatty acids in Arabidopsis thaliana leaves. Plant Biotechnology Journal. 2017;15:1010–23. Chisholm MJ, Hopkins CY. CONJUGATED FATTY ACIDS OF TRAGOPOGON AND CALENDULA SEED OILS. Can J Chem. 1960;38:2500–7. Holic R, Xu Y, Caldo KMP, Singer SD, Field CJ, Weselake RJ, et al. Bioactivity and biotechnological production of punicic acid. Appl Microbiol Biotechnol. 2018;102:3537–49. Sun H, Yang M, Gao Z, Wang X, Wu C, Wang Q, et al. Economic and environmental evaluation for a closed loop of crude glycerol bioconversion to biodiesel. Journal of Biotechnology. 2023;366:65–71. Bautista LF, Vicente G, Garre V. Biodiesel from microbial oil. Advances in Biodiesel Production [Internet]. Elsevier; 2012 [cited 2024 Jul 6]. p. 179–203. Available from: https://linkinghub.elsevier.com/retrieve/pii/B9780857091178500083 Gao Z, Ma Y, Wang Q, Zhang M, Wang J, Liu Y. Effect of crude glycerol impurities on lipid preparation by Rhodosporidium toruloides yeast 32489. Bioresource Technology. 2016;218:373–9. Terao J. Revisiting carotenoids as dietary antioxidants for human health and disease prevention. Food Funct. 2023;14:7799–824. Baccouch R, Shi Y, Vernay E, Mathelié-Guinlet M, Taib-Maamar N, Villette S, et al. The impact of lipid polyunsaturation on the physical and mechanical properties of lipid membranes. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2023;1865:184084. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4774339","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":336318328,"identity":"1026bd3e-3f14-45c2-9b88-cb2c556dea99","order_by":0,"name":"Daniela Krajciova","email":"","orcid":"","institution":"Institute of Animal Biochemistry and Genetics","correspondingAuthor":false,"prefix":"","firstName":"Daniela","middleName":"","lastName":"Krajciova","suffix":""},{"id":336318329,"identity":"e9e2713d-508c-4575-9d14-b36fd0b71256","order_by":1,"name":"Roman Holic","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYBAC+wYwdZiBj5mB8QGQlQDEzHi1GByAamFjZmA2IFELAwObBHFajncnfi5gOCzHxs78rPJHzeE8/gb2xwb4tNj3nN0sPYPhvzEbM5vZbZ5jh4slDvAYJ+DTYieRu0Gah+FwYhszD9ttBra0xIYDPMwH8Gkxlsjd/BumpfDHv7TE+QfYH+PVYjgjdxvcFgbeNpvEDQcY8DvM4MzZbdY8BodBfjGW5u2zKTY8zGOM1/sGx3s33+apOCzHz3/44ccf3yTy5I63P5bApwWqEZmDP1ZGwSgYBaNgFBADAEWYRcL25N5TAAAAAElFTkSuQmCC","orcid":"","institution":"Institute of Animal Biochemistry and Genetics","correspondingAuthor":true,"prefix":"","firstName":"Roman","middleName":"","lastName":"Holic","suffix":""}],"badges":[],"createdAt":"2024-07-20 18:44:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4774339/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4774339/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61987558,"identity":"b72ee0d2-b5cb-4d30-8345-3ee280906db3","added_by":"auto","created_at":"2024-08-08 01:46:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":780813,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence microscopy of \u003cem\u003eR. toruloides\u003c/em\u003e wild type IFO0880 strain.\u003cstrong\u003e \u003c/strong\u003eCells were grown in the rich medium (YPD) and in a lipid production medium (nitrogen limited MedA\u003csup\u003e+\u003c/sup\u003e) supplemented with 6% glucose or crude glycerol at 30°C for 48 h. Cells were stained with LD540 to visualize the lipid droplets. Scale bar: 10 µm. Abbreviations: BF, bright field.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4774339/v1/e4539dbddbe8284530ad168b.png"},{"id":61987280,"identity":"2ab8e768-dadb-4550-886c-03db7a6e9da5","added_by":"auto","created_at":"2024-08-08 01:38:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":53772,"visible":true,"origin":"","legend":"\u003cp\u003eScreening of \u003cem\u003eR. toruloides\u003c/em\u003e transformants for production of punicic acid (PuA).\u003cstrong\u003e \u003c/strong\u003eCells were grown in the lipid production MedA\u003csup\u003e+\u003c/sup\u003e medium containing 6% glucose at 30°C for 72 h. Abbreviations: TFA, total fatty acids.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4774339/v1/7e1efd1ca3f350d9ddf3bfea.png"},{"id":61987284,"identity":"4e5846d7-dccf-42bc-97c6-33f1fef8210d","added_by":"auto","created_at":"2024-08-08 01:38:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":111994,"visible":true,"origin":"","legend":"\u003cp\u003eFlask characterization of IFO0880 (wild type strain), PGI26 and PGI28 (P\u003csub\u003e\u003cem\u003ePGI1\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ePgFADX\u003c/em\u003e-T\u003csub\u003e\u003cem\u003ePMA1\u003c/em\u003e\u003c/sub\u003e), NAR13 and NAR16 (P\u003csub\u003e\u003cem\u003eNAR1\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ePgFADX\u003c/em\u003e-T\u003csub\u003e\u003cem\u003ePMA1\u003c/em\u003e\u003c/sub\u003e), PMA5 and PMA6 (P\u003csub\u003e\u003cem\u003ePMA1\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ePgFADX\u003c/em\u003e-T\u003csub\u003e\u003cem\u003ePMA1\u003c/em\u003e\u003c/sub\u003e) cultivated in MedA\u003csup\u003e+\u003c/sup\u003e with 6% glucose as the carbon source. \u003cstrong\u003eA)\u003c/strong\u003e Growth and glucose consumption, \u003cstrong\u003eB)\u003c/strong\u003e dry cell weight (DCW) and total fatty acid (TFA) production. Gray bar area represents TFA free biomass (g/L) and white area represents TFA (g/L).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4774339/v1/dac9c0f40f486580d35a711f.png"},{"id":61987282,"identity":"435ededa-d037-4989-a9dc-0b68ad2a531d","added_by":"auto","created_at":"2024-08-08 01:38:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":415681,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative TLC chromatography scans of \u003cstrong\u003eA)\u003c/strong\u003e polar lipids (phospholipids) and \u003cstrong\u003eB)\u003c/strong\u003e neutral lipids (NL) extracted from \u003cem\u003eR. toruloides\u003c/em\u003e wild type IFO0880 strain and strains expressing \u003cem\u003eP. granatum\u003c/em\u003e fatty acid conjugase (\u003cem\u003ePgFADX\u003c/em\u003e) grown for 168 h in MedA\u003csup\u003e+\u003c/sup\u003e supplemented with 6% glucose. 30 µg of pomegranate seed oil (PSO) was used. Relative fatty acid content in \u003cstrong\u003eC) \u003c/strong\u003ephospholipids and \u003cstrong\u003eD) \u003c/strong\u003etriacylglycerols. Abbreviations: CL, cardiolipin; ERG, ergosterol; FFA, free fatty acids; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PL, phospholipids: PS, phosphatidylserine; SE, steryl esters; TAG, triacylglycerols; TFA, total fatty acids.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4774339/v1/b215766b1e1101ecb7df7982.png"},{"id":61987285,"identity":"501d609a-f06f-4db2-a3c3-0ac55cd2e612","added_by":"auto","created_at":"2024-08-08 01:38:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":66032,"visible":true,"origin":"","legend":"\u003cp\u003eFlask characterization of IFO0880 (wild type strain), PGI26 and PGI28 (P\u003csub\u003e\u003cem\u003ePGI1\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ePgFADX\u003c/em\u003e-T\u003csub\u003e\u003cem\u003ePMA1\u003c/em\u003e\u003c/sub\u003e), NAR13 and NAR16 (P\u003csub\u003e\u003cem\u003eNAR1\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ePgFADX\u003c/em\u003e-T\u003csub\u003e\u003cem\u003ePMA1\u003c/em\u003e\u003c/sub\u003e), PMA5 and PMA6 (P\u003csub\u003e\u003cem\u003ePMA1\u003c/em\u003e\u003c/sub\u003e-\u003cem\u003ePgFADX\u003c/em\u003e-T\u003csub\u003e\u003cem\u003ePMA1\u003c/em\u003e\u003c/sub\u003e) cultivated in MedA\u003csup\u003e+\u003c/sup\u003e with 6% crude glycerol as the carbon source. \u003cstrong\u003eA)\u003c/strong\u003e Growth, \u003cstrong\u003eB)\u003c/strong\u003e biomass – dry cell weight (DCW) and total fatty acid (TFA) production. Gray area represents TFA free biomass (g/L) and white area represents TFA (g/L).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4774339/v1/47142445e5b29e587ac27b34.png"},{"id":61987286,"identity":"bcb6b544-8a16-43e7-bcf1-5be6a44481f5","added_by":"auto","created_at":"2024-08-08 01:38:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":432236,"visible":true,"origin":"","legend":"\u003cp\u003eTypical TLC chromatography scans of \u003cstrong\u003eA)\u003c/strong\u003e polar lipids (phospholipids) and \u003cstrong\u003eB)\u003c/strong\u003e neutral lipids (NL) extracted from \u003cem\u003eR. toruloides\u003c/em\u003e wild type IFO0880 strain and strains expressing \u003cem\u003eP. granatum\u003c/em\u003e fatty acid conjugase (PgFADX) grown for 168 h in MedA\u003csup\u003e+\u003c/sup\u003e supplemented with 6% crude glycerol. 30 ug of pomegranate seed oil (PSO) was used. Relative fatty acid content in \u003cstrong\u003eC) \u003c/strong\u003ephospholipids and \u003cstrong\u003eD) \u003c/strong\u003etriacylglycerols. Abbreviations: CL, cardiolipin; ERG, ergosterol;\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4774339/v1/ca0f0b5ece203265e713f617.png"},{"id":61987829,"identity":"fdab7711-4ab5-4f22-9d27-ba791e5ead96","added_by":"auto","created_at":"2024-08-08 01:54:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3225782,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4774339/v1/35d55c97-d1de-4f0a-86e4-acddf255765b.pdf"},{"id":61987281,"identity":"03cc4399-27f6-4153-9964-f5469fe5fe39","added_by":"auto","created_at":"2024-08-08 01:38:49","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":44554,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4774339/v1/5f2a5fa33ccb7e17170a3546.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The plasma membrane H+-ATPase promoter enables highly efficient production of punicic acid in Rhodotorula toruloides cultivated on glucose and crude glycerol","fulltext":[{"header":"Background","content":"\u003cp\u003eYeasts are commonly utilized in various biotechnological applications, including the production of biofuels, specialty chemicals, polymers, biomaterials, pharmaceuticals, enzymes, and recombinant proteins. They efficiently metabolize sugars as well as other carbon sources and, thanks to modern methods of genetic engineering, are sustainable, renewable, and have minimal environmental impact\u0026nbsp;[1].\u003c/p\u003e\n\u003cp\u003eThe oleaginous yeast \u003cem\u003eRhodotorula\u003c/em\u003e (\u003cem\u003eRhodosporidium\u003c/em\u003e) \u003cem\u003etoruloides\u003c/em\u003e is capable of high-density growth while simultaneously producing a high titer of lipids\u0026nbsp;[2]. It is also known as \u0026ldquo;the red yeast\u0026rdquo; due to its production of carotenoids, which give it its characteristic red coloration. Carotenoids, including beta-carotene, torulene, and torularhodin, are valuable for their antioxidant properties and their applications in the food, pharmaceutical, and cosmetic industries\u0026nbsp;[3,4]. In addition, \u003cem\u003eR. toruloides\u003c/em\u003e naturally utilizes various carbon sources for growth, including glucose, xylose, cellobiose, glycerol, acetic acid, and cellulose biomass hydrolysates\u0026nbsp;[5\u0026ndash;8], and exhibits halotolerance\u0026nbsp;[9]. Like other yeasts, significant lipid accumulation occurs under nitrogen-limited conditions\u0026nbsp;[10]. \u003cem\u003eR. toruloides\u003c/em\u003e has immense potential for the industrial production of value-added lipids. Every year, advances in molecular tools simplify genetic manipulations with this yeast, overcoming challenges in its utilization in both scientific research and industry\u0026nbsp;[11].\u003c/p\u003e\n\u003cp\u003ePunicic acid (PuA, C18:3Delta9cis,11trans,13cis) with three conjugated double bonds is an isomer of alpha-linolenic acid (C18:3Delta9cis,12cis,15cis), and it constitutes approximately 60-80% of pomegranate seed oil (PSO)\u0026nbsp;[12].\u0026nbsp;Oils enriched with conjugated fatty acids are valuable for their nutritional value and industrial applications.\u0026nbsp;Currently, significant attention is focused on the anticancer effects of PSO. It inhibits oxidation and prostaglandin synthesis, reduces the incidence of breast, prostate, and colon cancer, and increases the apoptosis of cancer cells\u0026nbsp;[13,14]. Recent studies also suggest that PuA could be used in the prevention and treatment of neurodegenerative diseases such as Alzheimer\u0026apos;s, Parkinson\u0026apos;s, and Huntington\u0026apos;s\u0026nbsp;[15].\u003c/p\u003e\n\u003cp\u003eIn pomegranate, PuA is formed by the activity of delta12-desaturase of oleic acid (FAD2) homologue, referred to as PgFADX (fatty acid conjugase)\u0026nbsp;[16,17]. This enzyme converts the delta12 double bond of linoleic acid (C18:2Delta9cis,12cis) within the phosphatidylcholine molecule into two conjugated double bonds at positions C11 and C13. PgFADX exhibits dual activity; it catalyzes the production of PuA and converts oleic acid into linoleic acid, the precursor of PuA. The heterologous expression of the \u003cem\u003ePgFADX\u003c/em\u003e in the \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e yeast has demonstrated that this yeast produces PuA in very limited amounts (0.8% of total fatty acids (TFA)) and only when grown in the presence of linoleic acid. Further efforts to enhance PuA production in recombinant \u003cem\u003eS. cerevisiae\u003c/em\u003e cells (a deletion mutant in the transcription factor Snf2p simultaneously expressing \u003cem\u003ePgFADX\u003c/em\u003e, \u003cem\u003ePgPDAT\u003c/em\u003e, and \u003cem\u003ePgLPCAT\u003c/em\u003e with the addition of 0.05% linoleic acid) led to a PuA accumulation reaching 3.37% of TFA\u0026nbsp;[18].\u003c/p\u003e\n\u003cp\u003eHeterologous expression of the \u003cem\u003ePgFADX\u003c/em\u003e in the \u003cem\u003eArabidopsis thaliana\u003c/em\u003e plant revealed that PgFADX inhibits the naturally occurring desaturase AtFAD2\u0026nbsp;[19]. An increased PuA accumulation was achieved in \u003cem\u003eA. thaliana\u003c/em\u003e when \u003cem\u003ePgFADX\u003c/em\u003e and \u003cem\u003ePgFAD2\u003c/em\u003e were simultaneously expressed in seed lines with a significantly elevated level of linoleic acid. Recently, in a modified strain of \u003cem\u003eBrassica napus\u003c/em\u003e with a higher oleic acid content, the co-expression of \u003cem\u003ePgFAD2\u003c/em\u003e and \u003cem\u003ePgFADX\u003c/em\u003e achieved 11.1% of PuA of TFA\u0026nbsp;[20]. The PuA distribution in recombinant plants was high in phospholipids compared to pomegranate and low in triacylglycerols (TAG). These results suggest that lipid metabolism regulation, substrate availability, and intracellular channeling of PuA from phospholipids to TAG are essential processes for effective PuA accumulation in pomegranate seeds.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInitial attempts to produce PuA in the fission yeast \u003cem\u003eSchizosaccharomyces pombe\u003c/em\u003e showed that, unlike the heterologous expression of \u003cem\u003ePgFAD2\u003c/em\u003e, yeasts which express \u003cem\u003ePgFADX\u003c/em\u003e exhibited slowed growth\u0026nbsp;[21]. Recombinant \u003cem\u003eS. pombe\u003c/em\u003e cells accumulated 38.7 mg/L of PuA, corresponding to 19.6% of PuA in TFA when \u003cem\u003ePgFADX\u003c/em\u003e was expressed, and 34.3 mg/L of PuA, corresponding to 25.1% of PuA in TFA, in the case of the co-expression of \u003cem\u003ePgFADX\u003c/em\u003e with \u003cem\u003ePgFAD2\u003c/em\u003e. The dynamics of PuA accumulation in these heterologous strains were different and correlated with the growth defects of the yeast cultures tested. This difference is likely due to an inefficient deposition of PuA into TAG, leading to PuA accumulation in phospholipids, as well as in the free fatty acid fraction. Accumulation of PuA in membrane lipids and free fatty acids can lead to PuA lipotoxicity, potentially resulting from its interference with essential cellular processes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn an effort to produce PuA by oleaginous organisms, recombinant strains of \u003cem\u003eYarrowia lipolytica\u003c/em\u003e were constructed\u0026nbsp;[22]. The promoter optimization for \u003cem\u003ePgFADX\u003c/em\u003e expression led to an improved PuA accumulation, from 0.9 to 1.8 mg/g of dry cell weight (DCW). The strain with the highest PuA production, expressing \u003cem\u003ePgFADX\u003c/em\u003e under the control of a strong erythritol-inducible promoter, accumulated 36.6 mg/L of PuA. A recent study demonstrated\u003cem\u003e\u0026nbsp;\u003c/em\u003ethat the yeast\u003cem\u003e\u0026nbsp;Y. lipolytica\u003c/em\u003e is a promising host for PuA production\u0026nbsp;[23]. The resulting production strain with\u0026nbsp;substantial multi-level genetic optimalization, such as an\u0026nbsp;increased linoleic acid content, multiple integrations of \u003cem\u003ePgFADX\u003c/em\u003e, blocked beta-oxidation, and several pathway modifications for acyl-chain editing, produced 100.6 mg/L of PuA (4.77% of TFA) in shake flask conditions, and 3072.72 mg/L of PuA in a fermenter.\u003c/p\u003e\n\u003cp\u003eIn this study, we constructed recombinant \u003cem\u003eR. toruloides\u003c/em\u003e strains for PuA production without disrupting the synthesis of carotenoids, which can prevent lipid oxidation during the downstream processes of PuA isolation. The effect of three different promoters and two carbon sources were tested. In the best PuA-producing strains cultivated in media containing 6% glucose and 6% waste crude glycerol, PuA accumulation reached 105.77 mg/L (6.06 mg/g DCW, 1.32% of TFA) and 72.81 mg/L (3.56 mg/g DCW, 0.68% of TFA), respectively. The levels achieved were comparable to those recently reported for engineered \u003cem\u003eY. lipolytica\u003c/em\u003e strains in flask experiments, even without substantial metabolic pathway modifications. This suggests that recombinant \u003cem\u003eR. toruloides\u003c/em\u003e is a more suitable oleaginous yeast for sustainable PuA production compared to \u003cem\u003eY. lipolytica\u003c/em\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eStrains, media and cultivation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStrains used in this study are listed in \u003cstrong\u003eTable 1\u003c/strong\u003e. NEB 5-alpha \u003cem\u003eEscherichia coli\u003c/em\u003e cells were used for plasmid construction. \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e EHA105 [24] were used for \u003cem\u003eA. tumefaciens\u003c/em\u003e-mediated transformation (ATMT) of \u003cem\u003eRhodotorula/Rhodosporidium toruloides\u003c/em\u003e. \u003cem\u003eR. toruloides\u003c/em\u003e IFO0880 (also known as APA2687 or NBRC0880) was a starting strain for all subsequent genetic modifications. \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eand \u003cem\u003eA. tumefaciens\u003c/em\u003e cells were cultivated at 37 \u0026deg;C and 30 \u0026deg;C in Luria Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl), respectively. To maintain the plasmids, the LB medium was supplemented with 50 mg/L kanamycin. \u003cem\u003eR. toruloides\u003c/em\u003e was cultivated at 30 \u0026deg;C, 160 rpm in the YPD medium (20 g/L glucose, 20 g/L peptone, and 10 g/L yeast extract). Cell growth was estimated spectrophotometrically (Spectrophotometer, Biochrom Libra S2, Cambridge, UK) by measuring the OD at 600 nm. The yeast inoculum was prepared in 5 mL of the YPD medium in a microbial tube and 18 h old inoculum was used for the inoculation of 50 mL production media MedA\u003csup\u003e+\u003c/sup\u003e to OD of 0.5 in a 250 mL baffled Erlenmeyer flask. The production MedA\u003csup\u003e+\u003c/sup\u003e growth medium was prepared by a modification of the MedA medium [25] to obtain a high C/N ratio leading to an increased accumulation of lipids in yeasts. The lipid production MedA\u003csup\u003e+\u003c/sup\u003e medium consisted of 1.5 g/L yeast extract, 0.5 g/L NH\u003csub\u003e4\u003c/sub\u003eCl, 7 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 5 g/L Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e.12H\u003csub\u003e2\u003c/sub\u003eO, 0.1 g/L CaCl\u003csub\u003e2\u003c/sub\u003e, 1.5 g/L MgSO\u003csub\u003e4\u003c/sub\u003e.7H2O, 10 mg/L ZnSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, 0.6 mg/L FeCl\u003csub\u003e3\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO, 0.07 mg/L MnSO\u003csub\u003e4\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003eO, 0.04 mg/L CuSO\u003csub\u003e4\u003c/sub\u003e.5H\u003csub\u003e2\u003c/sub\u003eO, and the carbon source, at a concentration of 60 g/L, was either glucose (Slavus, Bratislava, Slovakia) or crude glycerol (Mikrochem, Pezinok, Slovakia). For the nitrate reductase promoter, NH\u003csub\u003e4\u003c/sub\u003eCl was replaced by 0.8 g/L NaNO\u003csub\u003e3\u003c/sub\u003e in a MedA\u003csup\u003e+\u003c/sup\u003e medium. Cells were cultivated at 30 \u0026deg;C and 160 rpm inside an orbital shaker (Innova 40, Hamburg, Germany) for 24 to 168 h depending on the experiment. DCW was measured gravimetrically. The residual glucose in cell-free supernatant was determined using the GlucCell glucose monitoring system (Chemglass Life Sciences, New Jersey, USA), as previously reported [26].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Strains used in this study\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.75207986688852%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.302828618968388%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSource\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.75207986688852%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.302828618968388%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003eNEB 5-alpha\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.75207986688852%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003efhuA2\u0026Delta;(argF-lacZ)U169 phoA glnV44 \u0026Phi;80\u0026Delta;(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.302828618968388%\" valign=\"top\"\u003e\n \u003cp\u003eNEB\u0026nbsp;#C2987\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.75207986688852%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.302828618968388%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003eEHA105\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.75207986688852%\" valign=\"top\"\u003e\n \u003cp\u003ederivative of A281 (A136/pTiBo542)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.302828618968388%\" valign=\"top\"\u003e\n \u003cp\u003eSkerker J.M.,\u0026nbsp;\u003cbr\u003e\u0026nbsp;UC Berkeley\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003eEHA105-PGI1-PgFADX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.75207986688852%\" valign=\"top\"\u003e\n \u003cp\u003eEHA105/pZPK-P\u003cem\u003e\u003csub\u003ePGI1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ePgFADX\u003c/em\u003e-T\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e-P\u003cem\u003e\u003csub\u003eGPD1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003eHYG\u003c/em\u003e-T\u003cem\u003e\u003csub\u003eNOS\u003c/sub\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.302828618968388%\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003eEHA105-NAR1-PgFADX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.75207986688852%\" valign=\"top\"\u003e\n \u003cp\u003eEHA105/pZPK-P\u003cem\u003e\u003csub\u003eNAR1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ePgFADX\u003c/em\u003e-T\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e-P\u003cem\u003e\u003csub\u003eGPD1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003eHYG\u003c/em\u003e-T\u003cem\u003e\u003csub\u003eNOS\u003c/sub\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.302828618968388%\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003eEHA105-PMA1-PgFADX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.75207986688852%\" valign=\"top\"\u003e\n \u003cp\u003eEHA105/pZPK-P\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ePgFADX\u003c/em\u003e-T\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e-P\u003cem\u003e\u003csub\u003eGPD1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003eHYG\u003c/em\u003e-T\u003cem\u003e\u003csub\u003eNOS\u003c/sub\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.302828618968388%\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eRhodotorula toruloides\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.75207986688852%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.302828618968388%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003eIFO0880\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.75207986688852%\" valign=\"top\"\u003e\n \u003cp\u003eMAT A2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.302828618968388%\" valign=\"top\"\u003e\n \u003cp\u003eSkerker J.M.,\u0026nbsp;\u003cbr\u003e\u0026nbsp;UC Berkeley\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003ePGI4\u0026ndash;PGI36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.75207986688852%\" valign=\"top\"\u003e\n \u003cp\u003eIFO0880/P\u003cem\u003e\u003csub\u003ePGI1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ePgFADX\u003c/em\u003e-T\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e-P\u003cem\u003e\u003csub\u003eGPD1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003eHYG\u003c/em\u003e-T\u003cem\u003e\u003csub\u003eNOS\u003c/sub\u003e\u0026nbsp;\u003c/em\u003ecassette\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.302828618968388%\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003eNAR10\u0026ndash;NAR38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.75207986688852%\" valign=\"top\"\u003e\n \u003cp\u003eIFO0880/P\u003cem\u003e\u003csub\u003eNAR1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ePgFADX\u003c/em\u003e-T\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e-P\u003cem\u003e\u003csub\u003eGPD1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003eHYG\u003c/em\u003e-T\u003cem\u003e\u003csub\u003eNOS\u003c/sub\u003e\u0026nbsp;\u003c/em\u003ecassette\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.302828618968388%\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.945091514143094%\" valign=\"top\"\u003e\n \u003cp\u003ePMA1\u0026shy;\u0026shy;\u0026shy;\u0026shy;\u0026shy;\u0026ndash;PMA12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.75207986688852%\" valign=\"top\"\u003e\n \u003cp\u003eIFO0880/P\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ePgFADX\u003c/em\u003e-T\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e-P\u003cem\u003e\u003csub\u003eGPD1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003eHYG\u003c/em\u003e-T\u003cem\u003e\u003csub\u003eNOS\u003c/sub\u003e\u003c/em\u003e cassette\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.302828618968388%\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid construction, transformation, and verification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ePgFADX\u003c/em\u003e was codon-optimized according to the codon preference of \u003cem\u003eR. toruloides\u003c/em\u003e and synthetized by Generay Biotech (Shanghai, China). The terminator sequence of the plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPase (T\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e), the promoter sequences of glucose-6-phosphate isomerase (P\u003cem\u003e\u003csub\u003ePGI1\u003c/sub\u003e\u003c/em\u003e), the nitrate reductase (P\u003cem\u003e\u003csub\u003eNAR1\u003c/sub\u003e\u003c/em\u003e), and the plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPase (P\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e) were amplified from the genomic DNA of \u003cem\u003eR. toruloides\u003c/em\u003e using the primer pairs listed in \u003cstrong\u003eSupplementary\u003c/strong\u003e \u003cstrong\u003eTable S1\u003c/strong\u003e. The codon optimized \u003cem\u003ePgFADX\u003c/em\u003e sequence, the promoter and the terminator sequences are listed in \u003cstrong\u003eSupplementary\u003c/strong\u003e \u003cstrong\u003eTable S2\u003c/strong\u003e. To construct the plasmids pZPK-P\u003cem\u003e\u003csub\u003ePGI1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ePgFADX\u003c/em\u003e-T\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e-P\u003cem\u003e\u003csub\u003eGPD1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003eHYG\u003c/em\u003e-T\u003cem\u003e\u003csub\u003eNOS\u003c/sub\u003e\u003c/em\u003e, pZPK-P\u003cem\u003e\u003csub\u003eNAR1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ePgFADX\u003c/em\u003e-T\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e-P\u003cem\u003e\u003csub\u003eGPD1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003eHYG\u003c/em\u003e-T\u003cem\u003e\u003csub\u003eNOS\u003c/sub\u003e\u003c/em\u003e, and pZPK-P\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003ePgFADX\u003c/em\u003e-T\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e-P\u003cem\u003e\u003csub\u003eGPD1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003eHYG\u003c/em\u003e-T\u003cem\u003e\u003csub\u003eNOS,\u003c/sub\u003e\u003c/em\u003e three fragments consisting of the corresponding promoter sequence, \u003cem\u003ePgFADX\u003c/em\u003e, and T\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e were assembled into the binary linearized vector pZPK-P\u003cem\u003e\u003csub\u003eGPD1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003eHYG\u003c/em\u003e-T\u003cem\u003e\u003csub\u003eNOS\u003c/sub\u003e\u003c/em\u003e using the Gibson assembly reaction (E5520S, New England Biolabs, MA, USA). The plasmids were amplified in \u003cem\u003eE. coli\u003c/em\u003e, electroporated into \u003cem\u003eA. tumefaciens\u003c/em\u003e, and then used to transform \u003cem\u003eR. toruloides\u003c/em\u003e IFO0880 according to the ATMT, as described previously [27]. The transformants were resuspended in YPD and spread on YPD plates supplemented with 50 \u0026mu;g/mL hygromycin, 300 \u0026mu;g/mL cefotaxime, and 300 \u0026mu;g/mL carbenicillin to recover single clones. To verify the integration of the expected DNA fragment, \u003cem\u003eR. toruloides\u003c/em\u003e colonies were subjected to colony-PCR with the primers listed in \u003cstrong\u003eSupplementary Table S1\u003c/strong\u003e, according to the previously described method [28].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLipid extraction procedure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLipids were extracted using a method previously reported, with minor modifications\u0026nbsp;[21]. Briefly, yeast cells (aliquots of OD 50 - corresponding to approximately 10-13 mg of DCW) were collected by centrifugation, washed, and the cell pellets were frozen. The cells were suspended in 1 mL of a mixture of chloroform and methanol (2:1, v/v) containing the antioxidant butylated hydroxytoluene (BHT) at a final concentration of 0.01% and disrupted by FastPrep disintegrator (MP Biomedicals) with glass beads (diameter 0.4 mm, 3x40 s at the highest speed, with 5 min cooling on ice between cycles) to obtain homogenates. For the thin layer chromatography (TLC), lipids were extracted from homogenates by chloroform/methanol/water (1:2:0.8, v/v) and subsequently the proportion of the mixture was adjusted to 2:2:1.8 (v/v) at room temperature, according to the procedure of\u0026nbsp;[29]. The organic phase containing the lipids was separated by centrifugation; the lipids were dried under a stream of N\u003csub\u003e2,\u003c/sub\u003e and the dry lipids were dissolved in a 100 \u0026mu;L mixture of chloroform and methanol (2:1, v/v) and BHT, prior to use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalytical methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor TLC analyses, an aliquot of lipid extract corresponding to 0.5 mg of DCW for neutral lipids and 3 mg of DCW for phospholipids was applied to Silica Gel 60 TLC plates (Merck, Darmstadt, Germany) using a Linomat 5 semiautomatic sample applicator (CAMAG Linomat 5, Muttenz, Switzerland). Neutral lipids were separated by a two-step TLC solvent system using a method described previously (first step: petroleum ether/diethyl ether/acetic acid, 70:30:2; second step: petroleum ether and diethyl ether, 49:1)\u0026nbsp;[30]. Individual lipid spots were visualized by charring the plates, as previously reported\u0026nbsp;[31]. Phospholipids were separated by the solvent system (chloroform/methanol/acetic acid/water, 75:45:3:1), as described previously\u0026nbsp;[32]. Individual lipid spots were identified using lipid standards. The presence of PuA in individual lipids was determined by densitometry scan at 276 nm (CAMAG TLC Scanner 3, Muttenz, Switzerland).\u003c/p\u003e\n\u003cp\u003eFor fatty acid analysis, the total lipid homogenate, corresponding to approximately 10 mg DCW, was transmethylated with 5% Na-OCH\u003csub\u003e3\u003c/sub\u003e in methanol. Fatty acid methyl esters (FAME) were then extracted using \u003cem\u003en\u003c/em\u003e-hexane, as described previously [17]. The analysis of FAME was performed by the injection of 1 \u0026mu;L aliquots into a gas chromatography (GC) apparatus (GC2010Plus, Shimadzu) equipped with a BPX70 capillary column (30 m \u0026times; 0.25 mm \u0026times; 0.25 \u0026micro;m, SGE Analytical Science), as described previously [21,33]. Individual FAMEs were identified by comparing them with authentic standards (C4\u0026minus;C24 FAME mixture, Supelco). The quantification of individual fatty acids was conducted using tridecanoic acid methyl ester as an internal standard (Merck, Darmstadt, Germany). To determine the relative fatty acids content in TAG and phospholipids, the corresponding lipid spots were scraped off the TLC plate into glass tubes and transmethylated, as described above.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescence microscopy\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eR. toruloides w\u003c/em\u003eild type IFO0880 strain was cultured in a YPD media or in a MedA\u003csup\u003e+\u003c/sup\u003e media containing 6% glucose or glycerol for 48 h. The cell culture was diluted to OD = 2.0, harvested by centrifugation, washed once with a 50 mM Tris-HCl, pH 7.5, and suspended in 0.3 mL of 50 mM Tris-HCl, pH 7.5. LD540 (a stock solution of 0.05 mg/mL in ethanol) was added to the final concentration of 1 \u0026micro;g/mL, and the cells were incubated in the dark for 15 min at room temperature. A 3 \u0026micro;L drop of cell suspension was examined for the presence of the lipid droplets using a Leica DM5500 fluorescence microscope equipped with an HCX PL Fluotar 100\u0026times; objective, and a Leica DFC340 FX digital camera. Signals were detected using the filter system Y3 for CY3 green. All images were captured at the identical instrument settings and processed using LAS 3.0 software (Leica Microsystems, Wetzlar, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics and reproducibility\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were performed at least in duplicates. The data was expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors. All data analysis was performed by Excel.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003e\u003cem\u003ePromoters selection and strains construction\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe strain IFO0880 natively accumulates lipids at higher titers\u0026nbsp;[8]\u0026nbsp;and was therefore utilized for the engineering of PuA production. First, three types of plasmids based on the pZPK-P\u003cem\u003e\u003csub\u003eGPD1\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003eHYG\u003c/em\u003e-T\u003cem\u003e\u003csub\u003eNOS\u003c/sub\u003e\u003c/em\u003e backbone were constructed, with \u003cem\u003ePgFADX\u003c/em\u003e expression controlled by three different promoters. The choice of promoter is directly related to the expression level of the target protein [34,35]. An appropriately selected promoter can result in higher yields of the desired product without adversely affecting cell growth. So far, several promoters, including constitutive and inducible types, have been characterized in \u003cem\u003eR. toruloides\u003c/em\u003e. One of the promoters selected was a constitutive P\u003cem\u003e\u003csub\u003ePGI1\u003c/sub\u003e\u003c/em\u003e promoter of the glucose 6-phosphate isomerase. This promoter was previously shown to be four times stronger than the promoter of the glyceraldehyde 3-phosphate dehydrogenase, P\u003cem\u003e\u003csub\u003eGPD1\u003c/sub\u003e\u003c/em\u003e, when driving the expression of \u003cem\u003eHYG\u003c/em\u003e in cells supplemented with an increased concentration of hygromycin [36]. Since, in the effort to produce conjugated linolenic acid isomers (CLNA) in recombinant fission yeast, it was shown that a high accumulation of PuA and calendic acid influences cell growth [21,37], the second promoter selected was an inducible P\u003cem\u003e\u003csub\u003eNAR1\u003c/sub\u003e\u003c/em\u003e of the nitrate reductase regulated by the nitrogen source [38]. The third promoter used was P\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e of the plasma membrane proton-transporting ATPase, which is often employed as a constitutive promoter in the model yeast \u003cem\u003eS. cerevisiae\u003c/em\u003e. The expression cassettes containing codon optimized \u003cem\u003ePgFADX\u0026nbsp;\u003c/em\u003eunder the control of the three different selected promoters were randomly integrated into the \u003cem\u003eR. toruloides\u0026nbsp;\u003c/em\u003eIFO0880 genome using the ATMT. After selection of the stable transformants, the presence of an insertion cassette in the genomic DNA was confirmed by PCR analysis.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eScreening of the transformants expressing PgFADX\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eInitially, we analyzed whether \u003cem\u003eR. toruloides\u0026nbsp;\u003c/em\u003eaccumulates lipid storage organelles, lipid droplets, in a nitrogen-limited MedA\u003csup\u003e+\u003c/sup\u003e medium, which is used to stimulate the formation of lipids in oleaginous yeast \u003cem\u003eY. lipolytica\u0026nbsp;\u003c/em\u003e[39]. Lipid droplets were observed in living cells by staining with a lipid droplet-specific dye LD540. As shown in \u003cstrong\u003eFig. 1\u003c/strong\u003e, the \u003cem\u003eR. toruloides\u003c/em\u003e IFO0880 cultured in a MedA\u003csup\u003e+\u003c/sup\u003e medium showed enlarged lipid droplets compared to cells grown in a rich YPD medium. The presence of lipid droplets of bigger sizes was detected in cells grown in MedA\u003csup\u003e+\u003c/sup\u003e containing crude glycerol as a carbon source. Taken together, our results suggest that a MedA\u003csup\u003e+\u003c/sup\u003e medium supplemented with glucose or crude glycerol could be a suitable medium for the heterologous production of a single-cell oil containing PuA using metabolic engineering techniques.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSecondly, twelve randomly selected \u003cem\u003ePgFADX\u003c/em\u003e-containing transformants for each promoter were screened for their ability to produce PuA. This was because transformants obtained by the ATMT usually have the expression cassette randomly integrated into the genome, which can influence cell growth and the expression of the desired gene from the cassette [40]. As shown in \u003cstrong\u003eFig. 2\u003c/strong\u003e, randomly picked \u003cem\u003ePgFADX\u003c/em\u003e-containing transformants accumulated comparable levels of TFA per cell growth as wild type IFO0880. The most important result was the successful production of PuA in all tested engineered recombinant strains. Generally, a relatively higher content of PuA in TFA was observed for transformants expressing \u003cem\u003ePgFADX\u003c/em\u003e under the control of the P\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e promoter. The use of the P\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e promoter enhanced the production of PuA and the best PuA-producing strain, PMA5, accumulated 8.6-fold and 11.1-fold more \u0026micro;g/OD PuA compared to the PuA best-producing strains of the other two promoters, PGI28, and NAR13, respectively. This result confirmed the importance of promoter choice and suitable screening of engineered strains when ATMT transformation is used to obtain transformants with random integration of the desired cassette.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAccumulation and distribution of PuA in production media containing glucose\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIt was previously shown that the production of CLNA is a dynamic process [21,23,37]. Therefore, PuA-producing transformants were analyzed for the dynamics of PuA production in a time dependent manner. Two transformants for each promoter were selected, namely PGI26 and PGI28, NAR13 and NAR16, and PMA5 and PMA6, which express \u003cem\u003ePgFADX\u003c/em\u003e from P\u003cem\u003e\u003csub\u003ePGI\u003c/sub\u003e\u003c/em\u003e, P\u003cem\u003e\u003csub\u003eNAR1\u003c/sub\u003e\u003c/em\u003e, and P\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e promoters, respectively. First, the growth, biomass yield, glucose consumption, and fatty acid production in the engineered strains were compared to the wild type strain IFO0880 (\u003cstrong\u003eFig. 3\u0026nbsp;\u003c/strong\u003eand\u003cstrong\u003e\u0026nbsp;Table 2\u003c/strong\u003e). All the selected recombinant strains containing randomly integrated \u003cem\u003ePgFADX-\u003c/em\u003eexpression cassettes grew comparably well, with a slight increase in OD and biomass compared to the wild type strain. Most of the glucose was consumed after 72 h cultivation. All strains accumulated, on average, 40-50% of TFA in biomass. The fatty acid profile of the wild type strain remained stable during the cultivation periods of 72 h, 120 h, and 168 h (\u003cstrong\u003eSupplementary\u003c/strong\u003e \u003cstrong\u003eTable S3\u003c/strong\u003e). A substantial difference was observed in the ratio of monounsaturated to polyunsaturated fatty acid (MUFA/PUFA) for the PuA-producing recombinant strains. The expression of \u003cem\u003ePgFADX\u003c/em\u003e resulted in an increased relative content of oleic acid (C18:1) and decreased levels of linoleic acid (C18:2) and \u0026alpha;-linolenic acid (C18:3). These observed changes correlated with the elevation of PuA levels in the recombinant cells. This result suggests that the PgFADX might compete for the C18:1 substrate with the activity of the endogenous FAD2 in the engineered strains, similar to what observed in recombinant \u003cem\u003eArabidopsis thaliana\u003c/em\u003e plants [19].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe highest relative content of PuA reached 1.3% at 72 h and 120 h in the PMA6 strain containing the P\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e promoter, which was much higher than in the strains with \u003cem\u003ePgFADX\u003c/em\u003e expressed from the P\u003cem\u003e\u003csub\u003ePGI\u003c/sub\u003e\u003c/em\u003e and P\u003cem\u003e\u003csub\u003eNAR1\u003c/sub\u003e\u003c/em\u003e promoters (\u003cstrong\u003eTable 2\u003c/strong\u003e). When productivity is taken into consideration, in the best PuA-producing strain PMA6, the total PuA reached 105.8 mg/L and 6.1 mg/g DCW at 30\u0026deg;C\u0026nbsp;after 72 h (\u003cstrong\u003eTable 2\u003c/strong\u003e). Similar results were obtained with prolonged cultivation times of 120 h and 168 h, suggesting that the PuA level is stable over time in \u003cem\u003eR. toruloides\u0026nbsp;\u003c/em\u003ecells. The expression under P\u003cem\u003e\u003csub\u003ePGI\u003c/sub\u003e\u003c/em\u003e and P\u003cem\u003e\u003csub\u003eNAR1\u003c/sub\u003e\u003c/em\u003e promoters showed a 10-fold and 9-fold lower production of PuA, respectively, compared to the P\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e promoter.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e Fatty acid accumulation in strains cultivated in MedA\u003csup\u003e+\u003c/sup\u003e medium containing 6% glucose.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"572\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.947643979057592%\" valign=\"top\" style=\"width: 10.6643%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.518324607329843%\" valign=\"top\" style=\"width: 22.5525%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePgFADX\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003epromoter\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.202443280977313%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.136125654450261%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTFA (g/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.834205933682373%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTFA/DCW (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.263525305410122%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePuA (% of TFA)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.263525305410122%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePuA (mg/g DCW)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.834205933682373%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePuA (mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.947643979057592%\" rowspan=\"3\" valign=\"top\" style=\"width: 10.6643%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIFO0880\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.518324607329843%\" rowspan=\"3\" valign=\"top\" style=\"width: 22.5525%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.202443280977313%\" valign=\"top\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.136125654450261%\" valign=\"top\"\u003e\n \u003cp\u003e4.55 \u0026plusmn; 0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.834205933682373%\" valign=\"top\"\u003e\n \u003cp\u003e38.81 \u0026plusmn; 0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.263525305410122%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.263525305410122%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.834205933682373%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e120 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18%\" valign=\"top\"\u003e\n \u003cp\u003e6.20 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e41.80 \u0026plusmn; 1.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e168 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18%\" valign=\"top\"\u003e\n \u003cp\u003e6.61 \u0026plusmn; 0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e42.22 \u0026plusmn; 2.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.947643979057592%\" rowspan=\"3\" valign=\"top\" style=\"width: 10.6643%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePGI26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.518324607329843%\" rowspan=\"3\" valign=\"top\" style=\"width: 22.5525%;\"\u003e\n \u003cp\u003eP\u003cem\u003e\u003csub\u003ePGI1\u003c/sub\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.202443280977313%\" valign=\"top\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.136125654450261%\" valign=\"top\"\u003e\n \u003cp\u003e7.54 \u0026plusmn; 0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.834205933682373%\" valign=\"top\"\u003e\n \u003cp\u003e46.09 \u0026plusmn; 3.85\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.263525305410122%\" valign=\"top\"\u003e\n \u003cp\u003e0.13 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.263525305410122%\" valign=\"top\"\u003e\n \u003cp\u003e0.59 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.834205933682373%\" valign=\"top\"\u003e\n \u003cp\u003e9.74 \u0026plusmn; 0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e120 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18%\" valign=\"top\"\u003e\n \u003cp\u003e8.44 \u0026plusmn; 0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e45.23 \u0026plusmn; 1.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.12 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.55 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e10.35 \u0026plusmn; 0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e168 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18%\" valign=\"top\"\u003e\n \u003cp\u003e7.26 \u0026plusmn; 1.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e40.46 \u0026plusmn; 9.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.12 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.49 \u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e8.70 \u0026plusmn; 1.74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.947643979057592%\" rowspan=\"3\" valign=\"top\" style=\"width: 10.6643%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePGI28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.518324607329843%\" rowspan=\"3\" valign=\"top\" style=\"width: 22.5525%;\"\u003e\n \u003cp\u003eP\u003cem\u003e\u003csub\u003ePGI1\u003c/sub\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.202443280977313%\" valign=\"top\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.136125654450261%\" valign=\"top\"\u003e\n \u003cp\u003e6.76 \u0026plusmn; 0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.834205933682373%\" valign=\"top\"\u003e\n \u003cp\u003e37.97 \u0026plusmn; 1.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.263525305410122%\" valign=\"top\"\u003e\n \u003cp\u003e0.10 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.263525305410122%\" valign=\"top\"\u003e\n \u003cp\u003e0.37 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.834205933682373%\" valign=\"top\"\u003e\n \u003cp\u003e6.65 \u0026plusmn; 0.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e120 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18%\" valign=\"top\"\u003e\n \u003cp\u003e8.57 \u0026plusmn; 0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e45.05 \u0026plusmn; 2.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.10 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.43 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e8.21 \u0026plusmn; 0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e168 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18%\" valign=\"top\"\u003e\n \u003cp\u003e7.43 \u0026plusmn; 1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e39.71 \u0026plusmn; 5.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.09 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.37 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e6.90 \u0026plusmn; 0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.947643979057592%\" rowspan=\"3\" valign=\"top\" style=\"width: 10.6643%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNAR13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.518324607329843%\" rowspan=\"3\" valign=\"top\" style=\"width: 22.5525%;\"\u003e\n \u003cp\u003eP\u003cem\u003e\u003csub\u003eNAR1\u003c/sub\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.202443280977313%\" valign=\"top\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.136125654450261%\" valign=\"top\"\u003e\n \u003cp\u003e9.10 \u0026plusmn; 0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.834205933682373%\" valign=\"top\"\u003e\n \u003cp\u003e48.50 \u0026plusmn; 2.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.263525305410122%\" valign=\"top\"\u003e\n \u003cp\u003e0.13 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.263525305410122%\" valign=\"top\"\u003e\n \u003cp\u003e0.62 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.834205933682373%\" valign=\"top\"\u003e\n \u003cp\u003e11.62 \u0026plusmn; \u0026nbsp;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e120 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18%\" valign=\"top\"\u003e\n \u003cp\u003e8.69 \u0026plusmn; 0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e46.81 \u0026plusmn; 1.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.13 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.61 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e11.42 \u0026plusmn; 0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e168 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18%\" valign=\"top\"\u003e\n \u003cp\u003e8.75 \u0026plusmn; 0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e44.84 \u0026plusmn; 2.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.13 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.59 \u0026plusmn; 0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e11.42 \u0026plusmn; 1.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.947643979057592%\" rowspan=\"3\" valign=\"top\" style=\"width: 10.6643%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNAR16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.518324607329843%\" rowspan=\"3\" valign=\"top\" style=\"width: 22.5525%;\"\u003e\n \u003cp\u003eP\u003cem\u003e\u003csub\u003eNAR1\u003c/sub\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.202443280977313%\" valign=\"top\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.136125654450261%\" valign=\"top\"\u003e\n \u003cp\u003e9.27 \u0026plusmn; 0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.834205933682373%\" valign=\"top\"\u003e\n \u003cp\u003e46.53 \u0026plusmn; 0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.263525305410122%\" valign=\"top\"\u003e\n \u003cp\u003e0.07 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.263525305410122%\" valign=\"top\"\u003e\n \u003cp\u003e0.33 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.834205933682373%\" valign=\"top\"\u003e\n \u003cp\u003e6.59 \u0026plusmn; 0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e120 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18%\" valign=\"top\"\u003e\n \u003cp\u003e9.53 \u0026plusmn; 0.10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e49.86 \u0026plusmn; 2.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.07 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.36 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e6.97 \u0026plusmn; 0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e168 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18%\" valign=\"top\"\u003e\n \u003cp\u003e8.29 \u0026plusmn; 0.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e43.16 \u0026plusmn; 2.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.07 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.31 \u0026plusmn; 0.03\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e6.04 \u0026plusmn; 0.61\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.947643979057592%\" rowspan=\"3\" valign=\"top\" style=\"width: 10.6643%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePMA5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.518324607329843%\" rowspan=\"3\" valign=\"top\" style=\"width: 22.5525%;\"\u003e\n \u003cp\u003eP\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.202443280977313%\" valign=\"top\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.136125654450261%\" valign=\"top\"\u003e\n \u003cp\u003e7.35 \u0026plusmn; 0.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.834205933682373%\" valign=\"top\"\u003e\n \u003cp\u003e46.16 \u0026plusmn; 3.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.263525305410122%\" valign=\"top\"\u003e\n \u003cp\u003e0.97 \u0026plusmn; 0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.263525305410122%\" valign=\"top\"\u003e\n \u003cp\u003e4.45 \u0026plusmn; 0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.834205933682373%\" valign=\"top\"\u003e\n \u003cp\u003e70.91 \u0026plusmn; 2.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e120 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18%\" valign=\"top\"\u003e\n \u003cp\u003e7.39 \u0026plusmn; 0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e42.09 \u0026plusmn; 5.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.98 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e4.14 \u0026plusmn; 0.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e72.70 \u0026plusmn; 6.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e168 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18%\" valign=\"top\"\u003e\n \u003cp\u003e6.46 \u0026plusmn; 2.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e35.26 \u0026plusmn; 6.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e0.96 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e3.37 \u0026plusmn; 0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e61.63 \u0026plusmn; 19.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.947643979057592%\" rowspan=\"3\" valign=\"top\" style=\"width: 10.6643%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePMA6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.518324607329843%\" rowspan=\"3\" valign=\"top\" style=\"width: 22.5525%;\"\u003e\n \u003cp\u003eP\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.202443280977313%\" valign=\"top\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.136125654450261%\" valign=\"top\"\u003e\n \u003cp\u003e8.02 \u0026plusmn; 0.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.834205933682373%\" valign=\"top\"\u003e\n \u003cp\u003e45.98 \u0026plusmn; 2.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.263525305410122%\" valign=\"top\"\u003e\n \u003cp\u003e1.32 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.263525305410122%\" valign=\"top\"\u003e\n \u003cp\u003e6.06 \u0026plusmn; 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.834205933682373%\" valign=\"top\"\u003e\n \u003cp\u003e105.77 \u0026plusmn; 2.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e120 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18%\" valign=\"top\"\u003e\n \u003cp\u003e8.17 \u0026plusmn; 0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e44.05 \u0026plusmn; 1.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e1.27 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e5.60 \u0026plusmn; 0.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e103.90 \u0026plusmn; 4.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.444444444444445%\" valign=\"top\"\u003e\n \u003cp\u003e168 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18%\" valign=\"top\"\u003e\n \u003cp\u003e7.63 \u0026plusmn; 0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e42.39 \u0026plusmn; 5.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e1.25 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e5.28 \u0026plusmn; 0.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.88888888888889%\" valign=\"top\"\u003e\n \u003cp\u003e95.00 \u0026plusmn; 10.78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations: DCW, dry cell weight; PuA, punicic acid; TFA, total fatty acids.\u003c/p\u003e\n\u003cp\u003eIt is worth emphasizing that the PuA yield was 5.4-fold higher than the yield obtained in recombinant \u003cem\u003eS. pombe\u003c/em\u003e over-expressing \u003cem\u003ePgFADX\u003c/em\u003e from the strong inducible \u003cem\u003eNMT1\u003c/em\u003e promoter [21], and 2.9-fold higher than in the recombinant obese \u003cem\u003eY. lipolytica\u003c/em\u003e strain expressing \u003cem\u003ePgFADX\u0026nbsp;\u003c/em\u003efrom the hybrid inducible \u003cem\u003epEYK1 4AB-coreTEF\u003c/em\u003e promoter [22]. It is proposed that the main limitation in PuA accumulation is the inefficient flux of PuA from phospholipids to TAG in recombinant yeasts. This hypothesis was proven in the recombinant model yeast \u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003e[18] and more recently in the recombinant oleaginous yeast \u003cem\u003eY. lipolytica\u003c/em\u003e, which can accumulate increased levels of PuA only after significant multi-level genetic optimalization, including an improved supply of C18:2, expressing multiple copies of \u003cem\u003ePgFADX\u003c/em\u003e, the acyl-editing, \u0026beta;-oxidation, and glycerol-3-phosphate synthesis pathways [23].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo sum up, our results demonstrated that the recombinant oleaginous red yeast \u003cem\u003eR. toruloides,\u0026nbsp;\u003c/em\u003eexpressing \u003cem\u003ePgFADX\u0026nbsp;\u003c/em\u003efrom a P\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e promoter, is capable of producing PuA yields comparable to those recently reported for the engineered oleaginous yeast \u003cem\u003eY. lipolytica,\u003c/em\u003e with comprehensive genetic refinement. The relative level of PuA in engineered \u003cem\u003eR. toruloides\u0026nbsp;\u003c/em\u003eis not high; therefore, it is expected that further optimization of the metabolic pathways, including but not limited to the carbon and C18:2 supply, enhanced PuA synthesis, and PuA channeling to TAG lipid structures, could lead to a significant increase in PuA titer. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCLNA are preferentially synthesized through\u003cem\u003e\u0026nbsp;\u003c/em\u003ebiotransformation of linoleic acid esterified to phosphatidylcholine in native producers and then very efficiently channeled from membrane phospholipids to lipid storage depots, lipid droplets, in the form of TAG [19,41]. However, the precise mechanism of CLNA channeling is still not well understood and is currently under investigation. Previous studies have demonstrated a significant difference in the relative content of PuA in TAG and phosphatidylcholine lipid structures between pomegranate seeds, which naturally produce PuA, and the seeds of transgenic plants [19]. Therefore, the relative content of PuA in TAG and phospholipids was examined.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn order to characterize the distribution of PuA in individual lipid classes, the presence of conjugated double bonds in the PuA structure, which allows its detection under UV light, was utilized [42]. First, total lipid extracts of \u003cem\u003eR. toruloides\u003c/em\u003e were separated on TLC plates to analyze the phospholipids. With this approach, PuA was mainly detected in the phosphatidylcholine (\u003cstrong\u003eFig. 4A\u003c/strong\u003e), which is reported as the primary place for CLNA synthesis. Second, the total lipid extracts were loaded on a TLC plate and the plate was developed in conditions favoring the separation of neutral lipids. The majority of PuA was detected in TAG (\u003cstrong\u003eFig. 4B\u003c/strong\u003e). The presence of PuA in steryl esters could not be verified due to possible interference from the signal of some sterol molecules containing conjugated double bonds. Furthermore, PuA in a pool of free fatty acids was negligible. This is in contrast with the presence of CLNA in lipid extracts from recombinant yeast strains accumulating substantial amounts of free CLNA [22,37,43].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is worth noting that, although the relative amount of PuA in engineered \u003cem\u003eR. toruloides\u0026nbsp;\u003c/em\u003estrains is not high, the UV signal indicates that PuA is predominantly distributed in TAG lipid structures (\u003cstrong\u003eFig. 4A\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003eB\u003c/strong\u003e). This result suggests that, in engineered \u003cem\u003eR. toruloides\u0026nbsp;\u003c/em\u003estrains, the PuA is efficiently channeled from the site of synthesis (phosphatidylcholine) to the TAG lipid structures. However, this might be due to the very high ratio of TAG to phospholipids in engineered strains under tested conditions. To further analyze the distribution of PuA in lipid fractions in the two best PuA-producing strains, PMA5 and PMA6, the relative fatty acid content in phospholipids and TAG was determined (\u003cstrong\u003eFig. 4C\u003c/strong\u003e and \u003cstrong\u003e4D\u003c/strong\u003e). In both strains, the relative content of C18:1 increased in the lipid fractions examined compared to the wild-type strain, while the relative content of C18:3 showed an almost 4-fold decrease. The PuA contents in both strains were below 0.1% and approximately 1% in the phospholipids and TAG fractions, respectively. The high enrichment of PuA in the TAG lipid structures and, at the same time, the high yield of PuA with a minimal requirement for genome modification suggests that the red yeast \u003cem\u003eR. toruloides\u003c/em\u003e is a more suitable oleaginous yeast for PuA production than the yeast \u003cem\u003eY. lipolytica\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAccumulation and distribution of PuA in production media containing crude glycerol\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA byproduct of biodiesel production, a crude glycerol can be utilized for microbial lipid production using the yeast \u003cem\u003eR. toruloides\u0026nbsp;\u003c/em\u003e[5,44]. Converting a low-cost carbon source into a value-added product, such as PuA, could significantly reduce the upstream expenses, thereby making the overall production process more economically viable. In our initial experiments, the accumulation of enlarged lipid droplets was observed in the \u003cem\u003eR. toruloides\u0026nbsp;\u003c/em\u003ewild type IFO0880 strain, grown in a MedA\u003csup\u003e+\u003c/sup\u003e medium supplemented with 6% crude glycerol (\u003cstrong\u003eFig. 1\u003c/strong\u003e). Since the response of promoters to different carbon source may impact the final yield of the desired product\u0026nbsp;[34,35]\u0026nbsp;the PuA production efficiency, with the glucose replaced with crude glycerol, was evaluated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe recombinant strains producing the highest PuA yields for all three promoters were examined for growth, TFA accumulation, PuA content and its distribution. The procedure was similar to that used with the MedA\u003csup\u003e+\u003c/sup\u003e medium supplemented with glucose. As shown on \u003cstrong\u003eFig. 5A\u003c/strong\u003e, the growth of PuA-producing \u003cem\u003eR. toruloides\u003c/em\u003e strains was comparable to the growth of the wild type IFO0880. Compared to the glucose-containing medium (\u003cstrong\u003eFig. 3\u003c/strong\u003e), the growth in crude glycerol was slightly slower, but after 120 h, the cells reached a similar OD as when grown in glucose. The biomass yield and lipid accumulation increased with cultivation time (\u003cstrong\u003eFig. 5B\u003c/strong\u003e) and strains accumulated, on average, approximately 40-50% of TFA in biomass at the 120 h and 168 h time points (\u003cstrong\u003eTable\u0026nbsp;\u003c/strong\u003e3\u003cstrong\u003e)\u003c/strong\u003e. This result confirms that crude glycerol is a suitable low-cost carbon source for microbial lipid production using \u003cem\u003eR. toruloides\u003c/em\u003e. Similar to the glucose medium, the relative content of fatty acids in the wild type strain remained stable during the cultivation periods of 72 h, 120 h, and 168 h (\u003cstrong\u003eSupplementary\u003c/strong\u003e \u003cstrong\u003eTable S4\u003c/strong\u003e). However, the relative content of PUFA decreased in the MedA\u003csup\u003e+\u003c/sup\u003e supplemented with glycerol compared to the glucose medium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn general, the amount of PuA increased with the cultivation time (\u003cstrong\u003eTable 3\u003c/strong\u003e). In comparison to the glucose medium, the PuA yield significantly increased in strains with \u003cem\u003ePgFADX\u003c/em\u003e expressed from the P\u003cem\u003e\u003csub\u003eNAR1\u003c/sub\u003e\u003c/em\u003e promoter when cultivated on a low-cost crude glycerol medium. The highest relative level of PuA was 0.9% of TFA at 72 h in the PMA5 strain containing the P\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e promoter, which was again much higher than in the strains with \u003cem\u003ePgFADX\u003c/em\u003e expressed from the P\u003cem\u003e\u003csub\u003ePGI\u003c/sub\u003e\u003c/em\u003e and P\u003cem\u003e\u003csub\u003eNAR1\u003c/sub\u003e\u003c/em\u003e promoters. When productivity is taken into consideration, the best PuA-producing strain, PMA5, achieved a total PuA yield of 72.8 mg/L and 3.6 mg/g DCW at 30\u0026deg;C after 168 h in shake flask conditions. The level of PuA obtained is approximately two-fold higher than that has been reported for the engineered \u003cem\u003eS. pombe\u003c/em\u003e [21], and \u003cem\u003eY. lipolytica\u003c/em\u003e [22] strains. However, it is about one-third lower than the yields reported in a recent study on recombinant \u003cem\u003eY. lipolytica\u003c/em\u003e, which achieved higher yields through significant optimization of the metabolic pathways grown with glucose as a carbon source in shake flask conditions [23]. Nevertheless, it should be noted that, considering the upstream costs for the carbon source, the yield obtained from low-cost crude glycerol is more economically viable, despite the lower PuA levels achieved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3.\u0026nbsp;\u003c/strong\u003eFatty acid accumulation in strains cultivated in MedA\u003csup\u003e+\u003c/sup\u003e medium containing 6% crude glycerol.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"576\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.86159169550173%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eStrain\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.591695501730104%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003ePgFADX\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003epromoter\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131487889273357%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTFA (g/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTFA/DCW (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePuA (% of TFA)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePuA (mg/g DCW)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.262975778546714%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePuA (mg/L)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.86159169550173%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eIFO0880\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.591695501730104%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131487889273357%\" valign=\"top\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e5.26 \u0026plusmn; 0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e39.40 \u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.262975778546714%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.352422907488986%\" valign=\"top\"\u003e\n \u003cp\u003e120 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e9.19 \u0026plusmn; 0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.722466960352424%\" valign=\"top\"\u003e\n \u003cp\u003e48.73 \u0026plusmn; 0.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.704845814977972%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.352422907488986%\" valign=\"top\"\u003e\n \u003cp\u003e168 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e11.71 \u0026plusmn; 1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.722466960352424%\" valign=\"top\"\u003e\n \u003cp\u003e52.57 \u0026plusmn; 4.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.704845814977972%\" valign=\"top\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.86159169550173%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePGI26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.591695501730104%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eP\u003cem\u003e\u003csub\u003ePGI1\u003c/sub\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131487889273357%\" valign=\"top\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e4.92 \u0026plusmn; 0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e39.11 \u0026plusmn; 0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e0.04 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e0.17 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.262975778546714%\" valign=\"top\"\u003e\n \u003cp\u003e2.10 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.352422907488986%\" valign=\"top\"\u003e\n \u003cp\u003e120 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e8.43 \u0026plusmn; 0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.722466960352424%\" valign=\"top\"\u003e\n \u003cp\u003e46.38 \u0026plusmn; 0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.03 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.15 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.704845814977972%\" valign=\"top\"\u003e\n \u003cp\u003e2.76 \u0026plusmn; 0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.352422907488986%\" valign=\"top\"\u003e\n \u003cp\u003e168 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e8.84 \u0026plusmn; 1.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.722466960352424%\" valign=\"top\"\u003e\n \u003cp\u003e49.67 \u0026plusmn; 2.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.03 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.16 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.704845814977972%\" valign=\"top\"\u003e\n \u003cp\u003e2.93 \u0026plusmn; 0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.86159169550173%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePGI28\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.591695501730104%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eP\u003cem\u003e\u003csub\u003ePGI1\u003c/sub\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131487889273357%\" valign=\"top\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e4.85 \u0026plusmn; 0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e38.53 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e0.04 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e0.14 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.262975778546714%\" valign=\"top\"\u003e\n \u003cp\u003e1.73 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.352422907488986%\" valign=\"top\"\u003e\n \u003cp\u003e120 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e8.31 \u0026plusmn; 0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.722466960352424%\" valign=\"top\"\u003e\n \u003cp\u003e43.18 \u0026plusmn; 1.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.03 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.11 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.704845814977972%\" valign=\"top\"\u003e\n \u003cp\u003e2.14 \u0026plusmn; 0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.352422907488986%\" valign=\"top\"\u003e\n \u003cp\u003e168 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e9.82 \u0026plusmn; 2.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.722466960352424%\" valign=\"top\"\u003e\n \u003cp\u003e49.70 \u0026plusmn; 4.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.03 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.12 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.704845814977972%\" valign=\"top\"\u003e\n \u003cp\u003e2.44 \u0026plusmn; 0.57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.86159169550173%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNAR13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.591695501730104%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eP\u003cem\u003e\u003csub\u003eNAR1\u003c/sub\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131487889273357%\" valign=\"top\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e3.00 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e31.65 \u0026plusmn; 1.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e0.14 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e0.43 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.262975778546714%\" valign=\"top\"\u003e\n \u003cp\u003e4.05 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.352422907488986%\" valign=\"top\"\u003e\n \u003cp\u003e120 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e6.98 \u0026plusmn; 1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.722466960352424%\" valign=\"top\"\u003e\n \u003cp\u003e41.43 \u0026plusmn; 2.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.14 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.58 \u0026plusmn; 0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.704845814977972%\" valign=\"top\"\u003e\n \u003cp\u003e9.70 \u0026plusmn; 0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.352422907488986%\" valign=\"top\"\u003e\n \u003cp\u003e168 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e8.20 \u0026plusmn; 1.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.722466960352424%\" valign=\"top\"\u003e\n \u003cp\u003e50.20 \u0026plusmn; 4.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.26 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e1.26 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.704845814977972%\" valign=\"top\"\u003e\n \u003cp\u003e20.44 \u0026plusmn; 2.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.86159169550173%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNAR16\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.591695501730104%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eP\u003cem\u003e\u003csub\u003eNAR1\u003c/sub\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131487889273357%\" valign=\"top\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e3.16 \u0026plusmn; 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e33.71 \u0026plusmn; 0.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e0.09 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e0.29 \u0026plusmn; 0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.262975778546714%\" valign=\"top\"\u003e\n \u003cp\u003e2.68 \u0026plusmn; 0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.352422907488986%\" valign=\"top\"\u003e\n \u003cp\u003e120 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e7.88 \u0026plusmn; 1.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.722466960352424%\" valign=\"top\"\u003e\n \u003cp\u003e42.08 \u0026plusmn; 0.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.13 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.53 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.704845814977972%\" valign=\"top\"\u003e\n \u003cp\u003e9.91 \u0026plusmn; 1.87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.352422907488986%\" valign=\"top\"\u003e\n \u003cp\u003e168 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e8.79 \u0026plusmn; 1.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.722466960352424%\" valign=\"top\"\u003e\n \u003cp\u003e48.90 \u0026plusmn; 1.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.29 \u0026plusmn; 0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e1.40 \u0026plusmn; 0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.704845814977972%\" valign=\"top\"\u003e\n \u003cp\u003e24.98 \u0026plusmn; 2.69\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.86159169550173%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePMA5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.591695501730104%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eP\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131487889273357%\" valign=\"top\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e5.22 \u0026plusmn; 0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e37.33 \u0026plusmn; 1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e0.89 \u0026plusmn; 0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e3.33 \u0026plusmn; 0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.262975778546714%\" valign=\"top\"\u003e\n \u003cp\u003e46.54 \u0026plusmn; 2.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.352422907488986%\" valign=\"top\"\u003e\n \u003cp\u003e120 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e9.06 \u0026plusmn; 0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.722466960352424%\" valign=\"top\"\u003e\n \u003cp\u003e48.95 \u0026plusmn; 1.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.69 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e3.35 \u0026plusmn; 0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.704845814977972%\" valign=\"top\"\u003e\n \u003cp\u003e61.74 \u0026plusmn; 1.18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.352422907488986%\" valign=\"top\"\u003e\n \u003cp\u003e168 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e10.86 \u0026plusmn; 1.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.722466960352424%\" valign=\"top\"\u003e\n \u003cp\u003e52.83 \u0026plusmn; 1.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.68 \u0026plusmn; 0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e3.56 \u0026plusmn; 0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.704845814977972%\" valign=\"top\"\u003e\n \u003cp\u003e72.81 \u0026plusmn; 7.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.86159169550173%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePMA6\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.591695501730104%\" rowspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eP\u003cem\u003e\u003csub\u003ePMA1\u003c/sub\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.131487889273357%\" valign=\"top\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e5.17 \u0026plusmn; 0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.705882352941176%\" valign=\"top\"\u003e\n \u003cp\u003e34.88 \u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e0.75 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.14878892733564%\" valign=\"top\"\u003e\n \u003cp\u003e2.63 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.262975778546714%\" valign=\"top\"\u003e\n \u003cp\u003e38.94 \u0026plusmn; 0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.352422907488986%\" valign=\"top\"\u003e\n \u003cp\u003e120 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e8.46 \u0026plusmn; 0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.722466960352424%\" valign=\"top\"\u003e\n \u003cp\u003e41.64 \u0026plusmn; 2.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.60 \u0026plusmn; 0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e2.48 \u0026plusmn; 0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.704845814977972%\" valign=\"top\"\u003e\n \u003cp\u003e50.43 \u0026plusmn; 0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"10.352422907488986%\" valign=\"top\"\u003e\n \u003cp\u003e168 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e10.27 \u0026plusmn; 0.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.722466960352424%\" valign=\"top\"\u003e\n \u003cp\u003e47.96 \u0026plusmn; 2.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e0.61 \u0026plusmn; 0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.740088105726873%\" valign=\"top\"\u003e\n \u003cp\u003e2.92 \u0026plusmn; 0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.704845814977972%\" valign=\"top\"\u003e\n \u003cp\u003e62.44 \u0026plusmn; 3.56\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eAbbreviations: DCW, dry cell weight; PuA, punicic acid; TFA, total fatty acids.\u003c/p\u003e\n\u003cp\u003eTo examine the distribution of PuA in individual lipid classes, the total lipids were separated on TLC plates and the detection of PuA under UV light was used, as described above. Similarly, as with the glucose-containing medium, the signal for PuA was mainly detected in phosphatidylcholine (\u003cstrong\u003eFig. 6A\u003c/strong\u003e) and TAG lipid structures (\u003cstrong\u003eFig. 6B\u003c/strong\u003e). The signal for free PuA was negligible. Next, the relative contents of PuA\u0026nbsp;in phospholipid and TAG fractions in the two best PuA-producing strains, PMA5 and PMA6, was analyzed (\u003cstrong\u003eFig. 6C\u003c/strong\u003e and \u003cstrong\u003e6D\u003c/strong\u003e). The PuA content in both strains was approximately 0.2% and 0.6% in the phospholipids and TAG fractions, respectively. The enrichment of PuA in the single-cell oil, which are comprised of TAG lipid structures, was not as high as in the glucose medium. However, since the TAG are predominant lipid structures in \u003cem\u003eR. toruloides,\u0026nbsp;\u003c/em\u003egrown under nitrogen-limited conditions, with glycerol as the carbon source, the majority of PuA is stored in TAG lipid structures. Our results confirmed that the medium with a low-cost carbon source is suitable for the high production of PuA by the recombinant red yeast \u003cem\u003eR. toruloides\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe price of media components, mainly carbon and nitrogen sources, is a critical factor in any biotechnological process\u0026nbsp;[44,45]. \u003cem\u003eR. toruloides\u003c/em\u003e exhibits greater versatility than \u003cem\u003eY. lipolytica\u003c/em\u003e in assimilating low-cost carbon substrates, including crude glycerol, lignocellulosic hydrolysates, and molasses\u0026nbsp;[2]. This versatility significantly enhances its potential use in various biotechnological applications. Moreover, \u003cem\u003eR. toruloides\u003c/em\u003e can accumulate more lipids from crude glycerol than from pure glycerol, without being negatively affected by the impurities present in crude glycerol\u0026nbsp;[46]. An additional advantage of \u003cem\u003eR. toruloides\u0026nbsp;\u003c/em\u003eis its natural production of carotenoids, which act as antioxidants and are widely used in food, pharmaceuticals, and cosmetics\u0026nbsp;[3,4]. It has been demonstrated that carotenoids can effectively inhibit lipid peroxidation\u0026nbsp;[47]. Therefore, carotenoids, which naturally co-purify with lipids during the lipid extraction from engineered \u003cem\u003eR. toruloides\u003c/em\u003e cells (data not shown), could prevent PuA oxidation of PuA-enriched single cell oil.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnother notable difference between the two oleaginous yeasts is that \u003cem\u003eR. toruloides\u003c/em\u003e naturally synthesizes C18:3, unlike \u003cem\u003eY. lipolytica\u003c/em\u003e. It is well documented that the level of unsaturation in PUFA correlates with increased membrane fluidity, elasticity, and flexibility\u0026nbsp;[48]. Therefore, C18:3 has a greater impact on membrane properties compared to C18:2. It can be hypothesized that, due to this difference, \u003cem\u003eR. toruloides\u003c/em\u003e efficiently channels C18:3 from phospholipids to TAG lipid structures to maintain membrane properties. The presence of conjugated double bonds in PuA further impacts the cellular membrane properties. Considering these factors, along with the high accumulation of PuA in engineered \u003cem\u003eR. toruloides\u003c/em\u003e cells, it is evident that \u003cem\u003eR. toruloides\u003c/em\u003e may have even greater biotechnological potential for PuA production than \u003cem\u003eY. lipolytica\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eFor the first time, to our knowledge, the low-cost substrate, crude glycerol, was used for the production of a single-cell oil enriched in PuA using the recombinant red yeast \u003cem\u003eR. toruloides\u003c/em\u003e. The use of a promoter for plasma membrane ATPase for the expression of \u003cem\u003ePgFADX\u0026nbsp;\u003c/em\u003eimproved the overall productivity of the recombinant strain. The high PuA yield and its enrichment in TAG lipid structures, together with the minimal requirements necessary for genome modification, suggests that the red yeast \u003cem\u003eR. toruloides\u003c/em\u003e is a more suitable host for PuA production compared to \u003cem\u003eY. lipolytica\u003c/em\u003e. In addition, the red yeast naturally synthesizes carotenoids, which possess nutritional value, and can prevent oxidation of the CLNA during the downstream processing of lipids. With the further optimalization of the metabolic pathways, and medium formulation, further improvements of red yeast \u003cem\u003eR. toruloides\u003c/em\u003e for sustainable PuA production are expected.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank to Gina Geiselman (Sandia National Laboratories, Livermore, USA) for providing a detailed ATMT transformation protocol of \u003cem\u003eR. toruloides\u003c/em\u003e, Milan Certik and Peter Gajdos (Slovak Technical University, Bratislava, Slovakia) for providing the codon optimized \u003cem\u003ePgFADX\u003c/em\u003e sequence,\u0026nbsp;Jeffrey Michael Skerker (University of California, Berkeley, US), and Zongbao Kent Zhao (Dalian University of Technology, Dalian, China)\u0026nbsp;for kindly providing strains and cloning vector used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the Slovak Research and Development Agency under the contract No. APVV-20-0166\u0026nbsp;and the Grant Programme for SAS PhD students (APP0521).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDK:Methodology, Investigation, Writing – review, Funding acquisition. RH:Visualization, Supervision, Writing – original draft, review \u0026amp; editing, Funding acquisition. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVillena GK, Lude\u0026ntilde;a Y, Samolski I. Applications of yeast for environmental clean-up and sustainable agriculture. Advances in Yeast Biotechnology for Biofuels and Sustainability [Internet]. Elsevier; 2023 [cited 2024 Jun 26]. p. 193\u0026ndash;218. Available from: https://linkinghub.elsevier.com/retrieve/pii/B9780323954495000187\u003c/li\u003e\n\u003cli\u003ePark Y-K, Nicaud J-M, Ledesma-Amaro R. The Engineering Potential of Rhodosporidium toruloides as a Workhorse for Biotechnological Applications. Trends in Biotechnology. 2018;36:304\u0026ndash;17. \u003c/li\u003e\n\u003cli\u003eNabi F, Arain MA, Rajput N, Alagawany M, Soomro J, Umer M, et al. Health benefits of carotenoids and potential application in poultry industry: A review. Animal Physiology Nutrition. 2020;104:1809\u0026ndash;18. \u003c/li\u003e\n\u003cli\u003eZheng X, Hu R, Chen D, Chen J, He W, Huang L, et al. Lipid and carotenoid production by the Rhodosporidium toruloides mutant in cane molasses. Bioresource Technology. 2021;326:124816. \u003c/li\u003e\n\u003cli\u003eBommareddy RR, Sabra W, Maheshwari G, Zeng A-P. Metabolic network analysis and experimental study of lipid production in Rhodosporidium toruloides grown on single and mixed substrates. Microb Cell Fact. 2015;14:36. \u003c/li\u003e\n\u003cli\u003eHuang X-F, Liu J-N, Lu L-J, Peng K-M, Yang G-X, Liu J. Culture strategies for lipid production using acetic acid as sole carbon source by Rhodosporidium toruloides. Bioresource Technology. 2016;206:141\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eXu J, Zhao X, Wang W, Du W, Liu D. Microbial conversion of biodiesel byproduct glycerol to triacylglycerols by oleaginous yeast Rhodosporidium toruloides and the individual effect of some impurities on lipid production. Biochemical Engineering Journal. 2012;65:30\u0026ndash;6. \u003c/li\u003e\n\u003cli\u003eZhang S, Skerker JM, Rutter CD, Maurer MJ, Arkin AP, Rao CV. Engineering \u003cem\u003eRhodosporidium toruloides\u003c/em\u003e for increased lipid production. Biotech \u0026amp; Bioengineering. 2016;113:1056\u0026ndash;66. \u003c/li\u003e\n\u003cli\u003eTchakouteu SS, Kopsahelis N, Chatzifragkou A, Kalantzi O, Stoforos NG, Koutinas AA, et al. \u003cem\u003eRhodosporidium toruloides\u003c/em\u003e cultivated in NaCl‐enriched glucose‐based media: Adaptation dynamics and lipid production. Engineering in Life Sciences. 2017;17:237\u0026ndash;48. \u003c/li\u003e\n\u003cli\u003eRatledge C, Wynn JP. The Biochemistry and Molecular Biology of Lipid Accumulation in Oleaginous Microorganisms. Advances in Applied Microbiology [Internet]. Elsevier; 2002 [cited 2024 Jun 28]. p. 1\u0026ndash;52. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0065216402510005\u003c/li\u003e\n\u003cli\u003eWen Z, Zhang S, Odoh CK, Jin M, Zhao ZK. \u003cem\u003eRhodosporidium toruloides\u003c/em\u003e - A potential red yeast chassis for lipids and beyond. FEMS Yeast Research. 2020;20:foaa038. \u003c/li\u003e\n\u003cli\u003eTakagi T, Itabashi Y. Occurrence of mixtures of geometrical isomers of conjugated octadecatrienoic acids in some seed oils: Analysis by open-tubular gas liquid chromatography and high performance liquid chromatography. Lipids. 1981;16:546\u0026ndash;51. \u003c/li\u003e\n\u003cli\u003eAruna P, Venkataramanamma D, Singh AK, Singh RP. Health Benefits of Punicic Acid: A Review. Comprehensive Reviews in Food Science and Food Safety. 2016;15:16\u0026ndash;27. \u003c/li\u003e\n\u003cli\u003eShabbir MA, Khan MR, Saeed M, Pasha I, Khalil AA, Siraj N. Punicic acid: A striking health substance to combat metabolic syndromes in humans. Lipids Health Dis. 2017;16:99. \u003c/li\u003e\n\u003cli\u003eGuerra-Vazquez CM, Martinez-Avila M, Guajardo-Flores D, Antunes-Ricardo M. Punicic Acid and Its Role in the Prevention of Neurological Disorders: A Review. Foods. 2022;11. \u003c/li\u003e\n\u003cli\u003eHornung E, Pernstich C, Feussner I. Formation of conjugated Delta11Delta13-double bonds by Delta12-linoleic acid (1,4)-acyl-lipid-desaturase in pomegranate seeds. Eur J Biochem. 2002;269:4852\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eIwabuchi M, Kohno-Murase J, Imamura J. Delta 12-oleate desaturase-related enzymes associated with formation of conjugated trans-delta 11, cis-delta 13 double bonds. J Biol Chem. 2003;278:4603\u0026ndash;10. \u003c/li\u003e\n\u003cli\u003eWang J, Xu Y, Holic R, Yu X, Singer SD, Chen G. Improving the Production of Punicic Acid in Baker\u0026rsquo;s Yeast by Engineering Genes in Acyl Channeling Processes and Adjusting Precursor Supply. J Agric Food Chem. 2021;69:9616\u0026ndash;24. \u003c/li\u003e\n\u003cli\u003eMietkiewska E, Miles R, Wickramarathna A, Sahibollah AF, Greer MS, Chen G, et al. Combined transgenic expression of Punica granatum conjugase (FADX) and FAD2 desaturase in high linoleic acid Arabidopsis thaliana mutant leads to increased accumulation of punicic acid. Planta. 2014;240:575\u0026ndash;83. \u003c/li\u003e\n\u003cli\u003eXu Y, Mietkiewska E, Shah S, Weselake RJ, Chen G. Punicic acid production in Brassica napus. Metabolic Engineering. 2020;62:20\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eGaraiova M, Mietkiewska E, Weselake RJ, Holic R. Metabolic engineering of Schizosaccharomyces pombe to produce punicic acid, a conjugated fatty acid with nutraceutic properties. Appl Microbiol Biotechnol. 2017; \u003c/li\u003e\n\u003cli\u003eUrbanikova V, Park Y-K, Krajciova D, Tachekort M, Certik M, Grigoras I, et al. Yarrowia lipolytica as a Platform for Punicic Acid Production. IJMS. 2023;24:8823. \u003c/li\u003e\n\u003cli\u003eWang K, Zhou Y, Cao L, Lin L, Ledesma-Amaro R, Ji X-J. Engineering \u003cem\u003eYarrowia lipolytica\u003c/em\u003e for Sustainable Production of the Pomegranate Seed Oil-Derived Punicic Acid. J Agric Food Chem. 2024;72:3088\u0026ndash;98. \u003c/li\u003e\n\u003cli\u003eHood EE, Gelvin SB, Melchers LS, Hoekema A. NewAgrobacterium helper plasmids for gene transfer to plants. Transgenic Research. 1993;2:208\u0026ndash;18. \u003c/li\u003e\n\u003cli\u003eHoldsworth JE, Veenhuis M, Ratledge C. Enzyme Activities in Oleaginous Yeasts Accumulating and Utilizing Exogenous or Endogenous Lipids. Microbiology. 1988;134:2907\u0026ndash;15. \u003c/li\u003e\n\u003cli\u003eRoop JI, Chang KC, Brem RB. Polygenic evolution of a sugar specialization trade-off in yeast. Nature. 2016;530:336\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eCoradetti ST, Pinel D, Geiselman GM, Ito M, Mondo SJ, Reilly MC, et al. Functional genomics of lipid metabolism in the oleaginous yeast Rhodosporidium toruloides. eLife. 2018;7:e32110. \u003c/li\u003e\n\u003cli\u003eLin X, Wang Y, Zhang S, Zhu Z, Zhou YJ, Yang F, et al. Functional integration of multiple genes into the genome of the oleaginous yeast \u003cem\u003eRhodosporidium toruloides\u003c/em\u003e. FEMS Yeast Res. 2014;14:547\u0026ndash;55. \u003c/li\u003e\n\u003cli\u003eBligh EG, Dyer WJ. A RAPID METHOD OF TOTAL LIPID EXTRACTION AND PURIFICATION. Can J Biochem Physiol. 1959;37:911\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eSpanova M, Czabany T, Zellnig G, Leitner E, Hapala I, Daum G. Effect of lipid particle biogenesis on the subcellular distribution of squalene in the yeast Saccharomyces cerevisiae. J Biol Chem. 2010;285:6127\u0026ndash;33. \u003c/li\u003e\n\u003cli\u003eGaraiova M, Zambojova V, Simova Z, Griac P, Hapala I. Squalene epoxidase as a target for manipulation of squalene levels in the yeast Saccharomyces cerevisiae. FEMS Yeast Res. 2014;14:310\u0026ndash;23. \u003c/li\u003e\n\u003cli\u003eGarner K, Hunt AN, Koster G, Somerharju P, Groves E, Li M, et al. Phosphatidylinositol transfer protein, cytoplasmic 1 (PITPNC1) binds and transfers phosphatidic acid. J Biol Chem. 2012;287:32263\u0026ndash;76. \u003c/li\u003e\n\u003cli\u003eMietkiewska E, Siloto RM, Dewald J, Shah S, Brindley DN, Weselake RJ. Lipins from plants are phosphatidate phosphatases that restore lipid synthesis in a pah1Delta mutant strain of Saccharomyces cerevisiae. FEBS J. 2011;278:764\u0026ndash;75. \u003c/li\u003e\n\u003cli\u003eHo P-W, Klein M, Futschik M, Nevoigt E. Glycerol positive promoters for tailored metabolic engineering of the yeast Saccharomyces cerevisiae. FEMS Yeast Research [Internet]. 2018 [cited 2024 Jun 27];18. Available from: https://academic.oup.com/femsyr/article/doi/10.1093/femsyr/foy019/4898018\u003c/li\u003e\n\u003cli\u003eVogl T, Kickenweiz T, Pitzer J, Sturmberger L, Weninger A, Biggs BW, et al. Engineered bidirectional promoters enable rapid multi-gene co-expression optimization. Nat Commun. 2018;9:3589. \u003c/li\u003e\n\u003cli\u003eWang Y, Lin X, Zhang S, Sun W, Ma S, Zhao ZK. Cloning and evaluation of different constitutive promoters in the oleaginous yeast \u003cem\u003eRhodosporidium toruloides\u003c/em\u003e. Yeast. 2016;33:99\u0026ndash;106. \u003c/li\u003e\n\u003cli\u003eGaraiova M, Hua Q, Holic R. Heterologous Production of Calendic Acid Naturally Found in \u003cem\u003eCalendula officinalis\u003c/em\u003e by Recombinant Fission Yeast. J Agric Food Chem. 2023;71:3842\u0026ndash;51. \u003c/li\u003e\n\u003cli\u003eJohns AMB, Love J, Aves SJ. Four Inducible Promoters for Controlled Gene Expression in the Oleaginous Yeast Rhodotorula toruloides. Front Microbiol. 2016;7:1666. \u003c/li\u003e\n\u003cli\u003eHambalko J, Gajdo\u0026scaron; P, Nicaud J-M, Ledesma-Amaro R, Tupec M, Pichov\u0026aacute; I, et al. Production of Long Chain Fatty Alcohols Found in Bumblebee Pheromones by Yarrowia lipolytica. Front Bioeng Biotechnol. 2021;8:593419. \u003c/li\u003e\n\u003cli\u003eLin X, Gao N, Liu S, Zhang S, Song S, Ji C, et al. Characterization the carotenoid productions and profiles of three Rhodosporidium toruloides mutants from Agrobacterium tumefaciens-mediated transformation. Yeast. 2017;34:335\u0026ndash;42. \u003c/li\u003e\n\u003cli\u003eYurchenko O, Shockey JM, Gidda SK, Silver MI, Chapman KD, Mullen RT, et al. Engineering the production of conjugated fatty acids in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e leaves. Plant Biotechnology Journal. 2017;15:1010\u0026ndash;23. \u003c/li\u003e\n\u003cli\u003eChisholm MJ, Hopkins CY. CONJUGATED FATTY ACIDS OF TRAGOPOGON AND CALENDULA SEED OILS. Can J Chem. 1960;38:2500\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003eHolic R, Xu Y, Caldo KMP, Singer SD, Field CJ, Weselake RJ, et al. Bioactivity and biotechnological production of punicic acid. Appl Microbiol Biotechnol. 2018;102:3537\u0026ndash;49. \u003c/li\u003e\n\u003cli\u003eSun H, Yang M, Gao Z, Wang X, Wu C, Wang Q, et al. Economic and environmental evaluation for a closed loop of crude glycerol bioconversion to biodiesel. Journal of Biotechnology. 2023;366:65\u0026ndash;71. \u003c/li\u003e\n\u003cli\u003eBautista LF, Vicente G, Garre V. Biodiesel from microbial oil. Advances in Biodiesel Production [Internet]. Elsevier; 2012 [cited 2024 Jul 6]. p. 179\u0026ndash;203. Available from: https://linkinghub.elsevier.com/retrieve/pii/B9780857091178500083\u003c/li\u003e\n\u003cli\u003eGao Z, Ma Y, Wang Q, Zhang M, Wang J, Liu Y. Effect of crude glycerol impurities on lipid preparation by Rhodosporidium toruloides yeast 32489. Bioresource Technology. 2016;218:373\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eTerao J. Revisiting carotenoids as dietary antioxidants for human health and disease prevention. Food Funct. 2023;14:7799\u0026ndash;824. \u003c/li\u003e\n\u003cli\u003eBaccouch R, Shi Y, Vernay E, Matheli\u0026eacute;-Guinlet M, Taib-Maamar N, Villette S, et al. The impact of lipid polyunsaturation on the physical and mechanical properties of lipid membranes. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2023;1865:184084. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Synthetic biology, Metabolic engineering, Rhodosporidium toruloides, conjugated fatty acids (CLNA), FADX, single-cell oil, lipid, crude glycerol","lastPublishedDoi":"10.21203/rs.3.rs-4774339/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4774339/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePunicic acid is a conjugated fatty acid with a wide-range of nutraceutical properties naturally present in pomegranate seed oil. To meet the rising demand for pomegranate seed oil, a single-cell oil enriched in punicic acid provides a sustainable biomass-derived alternative. This study describes the production of a punicic acid-enriched single-cell oil through the engineering of the red yeast \u003cem\u003eRhodotorula toruloides\u003c/em\u003e grown in glucose and a low-cost substrate, crude glycerol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe gene for \u003cem\u003ePunica granatum\u003c/em\u003e fatty acid conjugase, \u003cem\u003ePgFADX\u003c/em\u003e, was randomly integrated into the genome of \u003cem\u003eR. toruloides\u003c/em\u003e without disrupting the carotenoid synthesis.\u003cem\u003e \u003c/em\u003eIn shake flask studies, the effects of three promoters (P\u003csub\u003e\u003cem\u003ePGI1\u003c/em\u003e\u003c/sub\u003e, P\u003csub\u003e\u003cem\u003eNAR1\u003c/em\u003e\u003c/sub\u003e, and P\u003csub\u003e\u003cem\u003ePMA1\u003c/em\u003e\u003c/sub\u003e) on punicic acid production were evaluated. A punicic acid titer of 105.77 mg/L and 72.81 mg/L was obtained from engineered cells expressing \u003cem\u003ePgFADX\u003c/em\u003e from the P\u003csub\u003e\u003cem\u003ePMA1\u003c/em\u003e\u003c/sub\u003e promoter cultivated for 72 hours in glucose and for 168 hours in crude glycerol, respectively. Furthermore, the detailed lipid analysis revealed a high enrichment of punicic acid in the triacylglycerol lipid structures, even without substantial modifications to the metabolic pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis report demonstrates the high potential of \u003cem\u003eR. toruloides\u003c/em\u003e in the biotransformation of a low-cost substrate, crude glycerol, into a value-added products such as punicic acid. The findings support the feasibility of using engineered \u003cem\u003eR. toruloides\u003c/em\u003e as a sustainable and efficient platform for the production of punicic acid-enriched single-cell oil.\u003c/p\u003e","manuscriptTitle":"The plasma membrane H+-ATPase promoter enables highly efficient production of punicic acid in Rhodotorula toruloides cultivated on glucose and crude glycerol","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-08 01:38:45","doi":"10.21203/rs.3.rs-4774339/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"11e71e98-87b6-41a9-9cd4-4dac25503e24","owner":[],"postedDate":"August 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-08T01:38:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-08 01:38:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4774339","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4774339","identity":"rs-4774339","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}