Increased Accumulation of Squalene in Engineered Yarrowia lipolytica through Deletion of PEX10 and URE2
ABSTRACT
Squalene is a triterpenoid serving as an ingredient of various products in the food, cosmetic, pharmaceutical industries. The oleaginous yeast Yarrowia lipolytica offers enormous potential as a microbial chassis for the production of terpenoids, such as carotenoid, limonene, linalool, and farnesene, as the yeast provides ample storage space for hydrophobic products. Here, we present a metabolic design that allows the enhanced accumulation of squalene in Y. lipolytica. First, we improved squalene accumulation in Y. lipolytica by overexpressing the genes (ERG and HMG) coding for the mevalonate pathway enzymes. Second, we increased the production of lipid where squalene is accumulated by overexpressing DGA1 (encoding diacylglycerol acyltransferase) and deleting PEX10 (for peroxisomal membrane E3 ubiquitin ligase). Third, we deleted URE2 (coding for a transcriptional regulator in charge of nitrogen catabolite repression [NCR]) to induce lipid accumulation regardless of the carbon-to-nitrogen ratio in culture media. The resulting engineered Y. lipolytica exhibited a 115-fold higher squalene content (22.0 mg/g dry cell weight) than the parental strain. These results suggest that the biological function of Ure2p in Y. lipolytica is similar to that in Saccharomyces cerevisiae, and its deletion can be utilized to enhance the production of hydrophobic target products in oleaginous yeast strains.
IMPORTANCE This study demonstrated a novel strategy for increasing squalene production in Y. lipolytica. URE2, a bifunctional protein that is involved in both nitrogen catabolite repression and oxidative stress response, was identified and demonstrated correlation to squalene production. The data suggest that double deletion of PEX10 and URE2 can serve as a positive synergistic effect to help yeast cells in boosting squalene production. This discovery can be combined with other strategies to engineer cell factories to efficiently produce terpenoid in the future.
INTRODUCTION
Squalene is an acyclic triterpenoid and a precursor for the biosynthesis of sterols, steroids, ubiquinones, and hopanoids (1). Because of its unique properties, squalene is widely used in the cosmetic industry as an antioxidant. Squalene is also used as an emulsion for the delivery of vaccines and other therapeutic substances and as a protective and preventive agent in cancer treatment (2, 3). Currently, squalene is mainly isolated from the liver oil of deep-sea sharks and plant oils (2). However, owing to the increasing global demand for squalene and concerns about the protection of marine animals and the fate of food crops, alternative production of squalene by microbial fermentation has drawn much attention as a sustainable source (4). So far, squalene production has been improved in many microorganisms. Common strategies in these works were engineering the methylerythritol-4-phosphate (MEP) or mevalonate (MVA) pathway to increase precursor pools and metabolic flux toward squalene and optimization of fermentation processes for increased accumulation of squalene. For example, the squalene content and volumetric titer of Aurantiochytrium sp. strain 18 W-13a increased to 171 mg/g dry cell weight (DCW) and 0.9 g/liter via optimization of fermentation conditions (5). In another study, Pseudozyma sp. strain JCC207 was identified and shown to produce up to 340.52 mg/liter squalene (6), and Escherichia coli accumulated squalene up to 230 mg/liter or 55 mg/g DCW by coexpressing a chimeric mevalonate pathway with human or Thermosynechococcus squalene synthase (1, 6–8). The squalene production by engineered Saccharomyces cerevisiae increased to 350 mg/g when the entire squalene biosynthesis pathway was overexpressed (9). The squalene accumulation in yeast mainly depended on the flux of the MVA pathway, downregulation of squalene degradation, and increasing the capacity of yeast to store the accumulated squalene in lipid droplets (10–12). Recently, we demonstrated that the increased lipid content of S. cerevisiae, providing storage for squalene accumulation, led to a 250-fold increase in squalene production compared to a parental strain (12). However, the limited storage capacity might still be a bottleneck to accommodate squalene in S. cerevisiae due to the restricted intracellular lipophilic storage space.
The oleaginous yeast Yarrowia lipolytica is an attractive host for the industrial production of hydrophobic compounds such as terpenoids due to its available genetic tools, large intercellular pool size of acetyl-coenzyme A (CoA), and rapid-growth phenotypes to high densities with high lipid content. Indeed, efficient production of a wide range of hydrophobic products, including lycopene, β-carotene, farnesene, and limonene, has been demonstrated in Y. lipolytica (13–17). Prior studies reported that Y. lipolytica is capable of producing intracellular lipid bodies where large amounts of hydrophobic compounds can be accumulated. For instance, lycopene production could be improved through the manipulation of lipid body formation (15). Additionally, Gao et al. and Larroude et al. reported a positive correlation between β-carotene and lipid contents and resulting high-level production of β-carotene in Y. lipolytica (18, 19).
As wild-type Y. lipolytica can synthesize squalene, we sought a metabolic engineering strategy for the efficient production of squalene by engineered Y. lipolytica. Specifically, genetic perturbations resulting in enhancement of the endogenous upstream MVA pathway and improved lipid accumulation were employed (Fig. 1). Notably, we first characterized the biological function of URE2 in Y. lipolytica, which encodes a bifunctional protein that is involved in both nitrogen catabolite repression and oxidative stress response. Deletion of URE2 led to improved squalene production, and double-gene deletion of URE2 and PEX10 resulted in improved squalene productivity.
FIG 1
RESULTS
Identification of limiting reactions in the mevalonate pathway for the production of squalene by Y. lipolytica.
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is known to be a major rate-limiting enzyme in the mevalonate (MVA) pathway in yeasts. Many studies have shown that the overexpression of HMG1, coding for HMG-CoA reductase, or its truncated variant (tHMG1) resulted in enhanced terpenoid biosynthesis in yeast (20–22). Meanwhile, the integration of HMG1 into an engineered Y. lipolytica strains led to a 6.9-fold increase in lycopene content and an 18-fold increase in limonene content, respectively (15, 17). As the overexpression of endogenous HMG1 improved squalene biosynthesis in Y. lipolytica, putative overexpression gene targets eliciting more squalene production were screened using the HMG1-overexpressing Y. lipolytica strain H1209 (23).
ERG10, coding for acetoacetyl-CoA thiolase, catalyzing acetyl-CoA to form acetoacetyl-CoA in the MVA pathway, was first selected as an overexpression target to improve squalene production. ERG12, coding for mevalonate kinase, catalyzing mevalonate to mevalonate-5-phosphate, was also selected as an overexpression target, as the overexpression of ERG12 along with the overexpression of HMG1 resulted in a 112-fold increase in limonene content (17). ERG20 coding for geranyl/farnesyl diphosphate synthase, contributing to the formation of geranyl diphosphate (GPP) and farnesyl pyrophosphate (FPP), was also selected as FPP is a precursor of squalene. Lastly, ERG9 coding for squalene synthase, playing a crucial role in the condensation of two molecules of FPP into squalene, was selected, as the overexpression of ERG20 and ERG9 had been used to enhance the production of sesquiterpenoids and triterpenoids (20, 24, 25).
In our previous study, overexpression of endogenous HMG1 in the wild-type Y. lipolytica Po1f strain (obtained strain H1209) led to a 3-fold increase of squalene biosynthesis. To identify potential limiting steps of squalene biosynthesis in the MVA pathway, the H1209 strain was then transformed with an expression cassette for each of the abovementioned genes to evaluate its contribution to squalene accumulation with overexpressed HMG1. Production of squalene by the transformants with each plasmid was then measured (Fig. 2A). All the transformants showed significantly higher squalene levels than the control strain H1209. These results are consistent with other works, which overexpressed key enzymes in the MVA pathway and observed enhanced production of terpenoids in S. cerevisiae and Y. lipolytica. In particular, the transformants of the ERG9 and ERG20 overexpression cassettes showed 5-fold higher squalene levels than the control strain, H1209. Similarly, overexpression of ERG9 or ERG20 resulted in enhanced production of protopanaxadiol, which is a triterpenoid using squalene as a precursor, in Y. lipolytica (26). This suggests that ERG9 and ERG20 are ideal engineering targets for further improving triterpenoid production in Y. lipolytica.
FIG 2
Further optimization of the MVA pathway to improve squalene production.
While extra gene transformants had higher squalene contents than control strain H1209, we speculated that a combination of the overexpression targets would further enhance squalene production by enhancing metabolic flux through the MVA pathway. To achieve this, the ERG9 overexpression cassette was integrated into the genome of H1209 strains with additional ERG10, ERG12, and ERG20 cassettes separately to yield strains CXS05, CXS06, and CXS07, respectively. This combination effort further increased the production of squalene than the HMG1/ERG9 cooverexpression strain to a limited extent (Fig. 2). Our results demonstrate that overexpression of multiple genes can lead to increased squalene contents. Understanding which of these factors plays the largest role in determining the yields of squalene production will lead to further insights for metabolic engineering efforts.
Overexpression of DGA1 and deletion of PEX10 to increase lipid accumulation and squalene production.
DGAT, catalyzing the ultimate step in triglyceride synthesis, is a key enzyme for lipid accumulation (27). There have been many studies describing the role of DGAT on lipid overproduction in yeasts, including S. cerevisiae and Y. lipolytica (13, 28, 29). To improve the lipid content, DGA1, coding for DGAT, was overexpressed in Y. lipolytica. The plasmids p1269-DGA1 and p1269-ERG9DGA1 were integrated into strain H1209 to obtain strains CXS08 and CXS09, respectively. As shown in Fig. 2B, the amounts of squalene produced by the CXS08 and CXS09 strains were 2.32 mg/g DCW and 4.10 mg/g DCW, which were 2.9-fold and 5.1-fold, respectively, the levels for strain H1209. At the same time, the lipid contents of CXS08 and CXS09 were 24 mg/g DCW and 36 mg/DCW, which were 50% and 132% higher, respectively, than those for strain H1209 (Fig. 3C).
FIG 3
Squalene is an important precursor for ergosterol synthesis. However, in this study, the higher content of squalene did not result in higher ergosterol content (Fig. 3A). The reason might be the existence of other rate-limiting enzymes in the ergosterol synthetic pathway. With the improvement of squalene production, the total lipid content in the engineered strains increased gradually, with no obvious differences in lipid composition (Fig. 3C and D). These results demonstrated that squalene production correlates with lipid content.
PEX10 is involved in the import of peroxisomal matrix proteins and is related to peroxisome proliferation in Y. lipolytica. The inactivation of PEX10 led to high eicosapentaenoic acid (EPA) titer and might increase the yields of other desirable lipid-related products (14). As such, PEX10 was disrupted in the HMG1-overpressing strain. As shown in Fig. 2B, the production level of squalene by the PEX10 downregulating strain CXS12 increased nearly 9-fold compared to that of the H1209 strain.
Identification, function analysis, and disruption of URE2 in Y. lipolytica.
To further improve lipid content, we sought to identify potential regulators of lipid metabolism. URE2 encodes a bifunctional protein that is involved in nitrogen catabolite repression and oxidative stress response in S. cerevisiae (30–32). When optimal sources of nitrogen are available, Ure2p acts as a transcriptional corepressor and downregulates the expression of many genes involved in nitrogen utilization by inhibiting the GATA transcriptional activators Gln3p and Gat1p (33, 34). As the induction of lipogenesis in yeast requires nitrogen limitations (27, 35), we hypothesized that disruption of URE2 in Y. lipolytica benefits the overproduction of lipids and accumulation of squalene. A Y. lipolytica URE2 (YlURE2) homolog was identified by BLAST search using S. cerevisiae URE2 (ScURE2) as a query sequence. YlURE2 was annotated YALI0C03069p (GenBank accession no. CAG81684.1), with 333 amino acids, and it shares 58% protein sequence with ScURE2 (Fig. 4A). A phylogenetic tree was constructed to show the relationship between YlURE2 and other homologs of ScURE2 from different yeasts. The phylogenetic tree was divided into two clades, and YlURE2 was found in a separate clade and phylogenetically far from ScURE2 (Fig. 4B).
FIG 4
In S. cerevisiae, Ure2p is structurally homologous to glutathione S-transferases and is known to be involved in salt tolerance (31, 36–38). To examine the role of YlURE2 in salt tolerance, we examined the growth of wild-type and ΔURE2 mutant strains on media with different nitrogen sources (yeast-peptone-glucose [YPD], yeast nitrogen base [YNB]-ammonia, and YNB-glutamate) containing high levels of heavy-metal ions (CrCl3, LiCl, ZnSO4, and Na2MoO4). When YPD was used as a medium, the ΔURE2 mutant was sensitive to Cr(III), Li(I), and Mo(VI). However, the deletion of URE2 enhanced cells’ ability to withstand toxic concentrations of Zn(II) in all media (YPD, YNB-ammonia, and YNB-glutamate). Further, the mutant cells exhibited a clearer sensitive phenotype with Cr(III) in both YNB-ammonia and glutamate media (see Fig. S1 in the supplemental material). These experiments suggest that Y. lipolytica Ure2p possesses a protection function and plays a role in detoxifying heavy-metal ions.
To elucidate the effects of URE2 disruption on squalene production, YlURE2 was deleted in the HMG1-overexpressing strain H1209. The levels of squalene in URE2-deleted and HMG1-overexpressing strain CXS15 increased slightly compared to that of the H1209 strain. We also deleted both PEX10 and URE2 in the H1209 strain. As shown in Fig. 2B, the combination of PEX10 and URE2 resulted in higher squalene levels than that of the single-deletion mutants. This synergistic effect of PEX10 and URE2 deletion on squalene production resulted in 191.28 mg/liter squalene. When lipid contents in the deletion strains were measured (Fig. 3C), the lipid accumulation in CXS18 was 2.1-fold higher than that in H1209, indicating the deletions of PEX10 and URE2 benefit lipid biosynthesis.
To assess the influence of PEX10 and URE2 deletions on mRNA levels of the key enzymes involved in lipid metabolism, nitrogen metabolism, and the MVA pathway, quantitative reverse transcription-PCR (qRT-PCR) was performed with RNAs isolated from the wild-type Po1f and engineered strains, CXS12 (HMG1 ΔPEX10), CXS15 (HMG1 ΔURE2), and CXS18 (HMG1 ΔPEX10 ΔURE2), cultured for 36 h in YPD medium. In the PEX10-disrupted strains, mRNA levels of POX1-6, MFE1, and POT1, which are involved in fatty acid degradation, were upregulated significantly, whereas ACL1, ACL2, ACC1, FAS1, FAS2, and DAG1, which were associated with fatty acid biosynthesis, were downregulated, as shown in Fig. 5. Although the mRNA level of DAG2 showed considerable changes, which was consistent with the conclusion of Morin et al. (39), Dga1p is thought to act in concert with triglyceride lipase to balance TAG flux in and out of lipid bodies (40–42). In S. cerevisiae, Gln3p and Gat1p belong to GATA of transcription regulators, and Ure2p is considered a negative regulator of GATA-dependent transcription (43). mRNA levels of GLN3 and GAT1 increased in the URE2-deleted strains compared with the wild-type strain. This was in accordance with published results for S. cerevisiae (31). Notably, MEP2 coding for ammonium permease was upregulated 24.8-fold in the ΔURE2 mutant compared to the wild-type strain, suggesting that Ure2p represses the transcription of MEP2. The expression levels of HMG1 were increased in all the engineered strains; however, it was interesting to see a decrease in HMG1 transcripts in the iterative strains (CXS15 and CXS18).
FIG 5
Increased production of squalene with combinatorial genetic perturbations.
After identifying gene targets eliciting enhanced production of lipid and squalene in Y. lipolytica, we combined them to further improve squalene production. Thus, the plasmids that carried the DGA1 expression cassette and ERG9/DGA1 expression cassettes were introduced into the background CXS18 strain (overexpression of HMG1, deletion of URE2 and PEX10) to obtain strains CXS19 and CXS20. As shown in Fig. 2B, both strains produced more squalene than the CXS18 strain, and maximum squalene concentrations of 240.5 mg/liter and 22.0 mg/g were achieved by CXS19. The combination of genetic perturbations successfully maximized squalene production and at the same time incrementally increased lipid aggregation from 4.0% to 9.4% without substantial changes in the proportion of fatty acids (Fig. 3C and D). Indeed, these results are consistent with a previous report with S. cerevisiae, enhancing the accumulation of lipid facilitated the production of squalene (12). In yeast, squalene can be further metabolized to ergosterol, which constitutes the predominant sterols in the cell membrane and is necessary to yeast survival. The ergosterol concentrations were not significantly different among the various engineered strains, suggesting that the genetic perturbations are not less efficient in converting squalene into ergosterol in the present study.
DISCUSSION
Many metabolic engineering strategies have been employed to generate high terpenoid production in microbial hosts as a sustainable alternative to current production methods. Successful terpenoid production has been reported in the model microorganisms E. coli and S. cerevisiae through a multimodular strategy by engineering the MEP or MVA pathway and the redox cofactor supply modules and precursor production (e.g., acetyl-CoA) and restraining competing pathways and optimizing fermentation processes. In addition to the work on terpenoid production in model microorganisms, the potential of several other hosts, including the oleaginous yeast Y. lipolytica, has been demonstrated in recent years. In Y. lipolytica, successful efforts have been made to overexpress the structural genes in the MVA pathway for the improvement of terpenoid production. An effective strategy is the overexpression of HMG-CoA reductase, which is a rate-limiting enzyme in the MVA pathway. In S. cerevisiae, the overexpression of tHMG1 lacking a regulatory domain significantly improves terpenoid production (44, 45). When it came to Y. lipolytica, full-length HMG1 was overexpressed to boost lycopene, limonene, and linalool production (15, 17, 46). Kildegaard et al. compared the performance of complete and truncated HMG1 variants and demonstrated that the expression of the full-length HMG1 was more effective than truncated HMG1 in enhancing β-carotene production (47). In our previous study, the overexpression of endogenous HMG1 led to a significant increase in squalene production up to 3-fold higher than that of the wild-type strain. However, the overexpression of heterologous tHMG1 from S. cerevisiae impaired squalene accumulation in Y. lipolytica (23). For high-level squalene production, extensive engineering of the MVA pathway flux is then bolstered by simultaneous overexpression of HMG1 with other structural genes in the MVA pathway. In our study, the optimal combination was obtained from coexpression of ERG9 and ERG20 with HMG1, resulting in a 4.2-fold increase of squalene content compared to the HMG1-overexpressing strain and reaching 4.2 mg/g dry cell weight (DCW). To further increase the squalene productivity, enhancement of the HMG1 gene dosage may be considered, because it was reported that the β-carotene content of Y. lipolytica could be improved by optimizing the expression level of HMG1, which subsequently enhanced HMG-CoA catalytic activity (18). With the recently established CRISPR/Cas9 system, further optimization of the genetic tools for multicopy integration could be easily transferred to our metabolic engineering strategy for Y. lipolytica (48, 49).
Our previous studies on squalene production in S. cerevisiae have suggested the introduction of genetic perturbations known to increase lipid contents to enhance squalene accumulation, as the lipid body can store squalene. Another study found overexpressed fatty acid synthases FAS1/FAS2 led to a significant increase in squalene production (50). Other groups working on Y. lipolytica reported a positive correlation between carotenoid synthesis and lipid content (18, 19). Therefore, we speculated that the aggregation of lipid in Y. lipolytica could also contribute to squalene overproduction. To this end, we turn our attention to lipid accumulation. While there have been significant efforts to increase lipid production in Y. lipolytica and other yeasts, fewer efforts have been focused on engineering Y. lipolytica to improve lipid metabolism and enable increase squalene accumulation. In the present study, peroxisomes were impaired by deletion of the peroxisomal biogenesis gene PEX10 to increase lipid content and synergistically boosted squalene production in engineered Y. lipolytica. However, several research groups have targeted peroxisomes as dynamic depots for the storage of terpenoids by introducing a complete MVA pathway in peroxisomes and achieved high-level productivity of terpenoids in S. cerevisiae (9, 51). Future work will be dedicated to examining the compartmentalization strategy in Y. lipolytica for an enhanced production of acetyl-CoA derived chemicals.
In general, induction of lipid body accumulation in microorganisms, such as the oleaginous yeast, microalgae, and bacteria, is linked to nitrogen limitation (52, 53). Thus, it is tempting to hypothesize that nitrogen catabolite repression (NCR) (i.e., the genes involved in the utilization and transport of poor nitrogen sources are repressed in the presence of preferred nitrogen sources) is involved in coordinating lipid metabolism with growth. Several NCR transcription factors have been identified, including the GATA-family transcription activators Gln3p and Gat1p, the repressors Dal80p and Gzf3p, and the zinc finger transcription factors Dal81p and Dal82p (54, 55). Among these, Gln3p activity is inhibited by Ure2p, a negative nitrogen regulator that plays a central role in NCR in S. cerevisiae under lush, nitrogen-replete conditions. As nitrogen becomes limiting or rapamycin is added, TOR is inactivated; consequently, Gln3p relocates to the nucleus and depresses NCR-sensitive transcription (56–60). It has been assumed by several authors that inhibition of TOR by rapamycin mimics nitrogen starvation (61, 62). A more recent study conducted on S. cerevisiae showed that TOR inhibition results in fast lipid droplet accumulation with a lower growth rate, which, on the contrary, is inhibited by rapamycin in mammals (63, 64). Their result further demonstrated that Gln3p and Gat1p could regulate the full induction of lipid droplets by rapamycin (63). In this study, we investigated the regulatory roles of Ure2p in a naturally minimized genomic background (Fig. 5). The data from qRT-PCR showed that the GLN3 and GAT1 transcription levels were higher in ΔURE2 than WT strains in nitrogen-rich medium, which is consistent with previous findings in S. cerevisiae (31).
Nutrient restriction can induce lipogenesis with a significant reduction in cell growth in microorganisms. For economically feasible biobased lipid production, the metabolic uncoupling of cell growth from intracellular lipid accumulation is a prerequisite. Previous studies showed that higher rapamycin concentrations resulted in growth inhibition in S. cerevisiae. In our study, with the increase in lipid accumulation, no obvious differences in optical density (OD) between the set of ΔPEX10 ΔURE2 mutant strains were observed during cultivation, indicating that cell growth was not affected by the abolishment of the URE2 gene in the nitrogen-replete medium. This phenomenon is in striking contrast to the inhibition of TOR by supplementation with rapamycin on algae and S. cerevisiae growth, where lipid is aggregated with decreased biomass formation. A recent study demonstrated, for the first time, that lipogenesis could be induced with the addition of rapamycin in an oleaginous yeast, Trichosporon oleaginosus, without compromising growth (65). Although the structure and functions of TOR complexes, such as TORC1 activation of translation and transcription, are conserved from yeast to mammals, the downstream processes are vastly different depending on the organism (66). In Y. lipolytica, such regulation of lipid metabolism by TOR is not yet well established. We proposed a model of the URE2-related regulatory network in Y. lipolytica by combining the literature with our work (Fig. 6). However, to gain deeper knowledge of the TOR signaling pathway associated with lipogenesis, additional experiments are needed.
FIG 6
Taken together, our findings demonstrate the potential of the combination of the MVA pathway optimization and lipid metabolic engineering for the overproduction of squalene in oleaginous organisms. The discovery of the latent role of URE2 in squalene accumulation enabled a novel strategy to increase squalene production that can be combined with other strategies to engineer cell factories for the efficient production of squalene in the future. More research into how the TOR signaling pathway is associated with lipogenesis will yield additional insights into the control and induction of lipid accumulation in oil yeasts.
MATERIALS AND METHODS
Strains, media, and reagents.
The vectors and strains used in this study are listed in Tables 1 and 2, respectively. E. coli JM109 was used for cloning and plasmid propagation. JM109 was grown in Luria-Bertani (LB) medium supplemented with 100 mg/liter ampicillin or 50 mg/liter kanamycin for plasmid propagation at 37°C. Y. lipolytica strain Po1f (MATa leu2-270 ura3-302 xpr2-322, axp-2; ATCC no. MYA-2613) (67), a leucine and uracil auxotroph, was used as the base strain for all genome editing and expression experiments in this study. The Y. lipolytica strains were cultured and fermented in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) at 30°C and 220 rpm. All strains were first inoculated into 10-ml culture tubes containing 2 ml medium and incubated at 30°C at 220 rpm until the OD at 600 nm (OD600) reached 1.0. For the fermentation, the precultured Y. lipolytica strains were transferred to 250-ml shake flasks containing 50 ml YPD (initial OD600 of 0.01) for 5 days. All flask fermentation results represente means ± standard deviations (SD) from three independent experiments. The synthetic medium YNB (0.67% yeast nitrogen base without amino acids, 1% glucose, and 1.6% agar), lacking leucine and uracil where appropriate, was used for the screening of transformants. Plasmid extraction, gel extraction, and purification of DNA were conducted using assay kits from Sangon Biotech Co., Ltd. (Shanghai, China). Primer synthesis was conducted by GeneRay Biotech Co., Ltd. (Shanghai, China). Squalene and ergosterol standards were purchased from Sigma-Aldrich (St. Louis, MO).
TABLE 1
| Vector | Description | Source |
|---|---|---|
| JMP113 | 1.2-kb YlURA3 fragment in pBluescript II KS+ | 68 |
| pUB4-CRE | 2.1-kb Cre fragment in pUB4 | 68 |
| pINA1312 | Y. lipolytica integrative plasmid, hp4d promoter, XPR2 terminator, ura3d1 selection marker, Kmr | 71 |
| pINA1269 | Y. lipolytica integrative plasmid, hp4d promoter, XPR2 terminator, LEU2 selection marker, Ampr | 71 |
| p1312HMG1 | Constitutively expressed HMG1 gene | This study |
| p1269ERG10 | Constitutively expressed ERG10 gene | This study |
| p1269ERG12 | Constitutively expressed ERG12 gene | This study |
| p1269ERG9 | Constitutively expressed ERG9 gene | This study |
| p1269ERG20 | Constitutively expressed ERG20 gene | This study |
| p1269ERG9ERG10 | Constitutively expressed ERG9 and ERG10 genes | This study |
| p1269ERG9ERG12 | Constitutively expressed ERG9 and ERG12 genes | This study |
| p1269ERG9ERG20 | Constitutively expressed ERG9 and ERG20 genes | This study |
| p1269DGA1 | Constitutively expressed DGA1 gene | This study |
| p1269ERG9DGA1 | Constitutively expressed ERG9 and DGA1 genes | This study |
| pBluescript II SK+ | E. coli recombinant plasmid, Ampr | Stratagene |
| SK-PEX10 | PEX10 gene containing promoter and terminator in pBluescript II SK+ | This study |
| SK-PEX10-URA3 | Promoter of the PEX10 gene-URA3 gene, the terminator of the PEX10 gene in pBluescript II SK+ | This study |
| SK-URE2 | The URE2 gene containing promoter and terminator in pBluescript II SK+ | This study |
| SK-URE2-URA3 | The promoter of the URE2 gene-URA3 gene-the terminator of the URE2 gene in pBluescript II SK+ | This study |
TABLE 2
| Strains | Description | Source |
|---|---|---|
| Escherichia coli | ||
| JM109 | For construction of recombinant vectors | Invitrogen |
| Yarrowia lipolytica | ||
| MYA2613 (Po1f) | leu2 and ura3 mutant | ATCC |
| H1209 | Po1f cells harboring p1312HMG1 | This study |
| CXS01 | Po1f cells harboring p1312HMG1 and p1269ERG10 | This study |
| CXS02 | Po1f cells harboring p1312HMG1 and p1269ERG12 | This study |
| CXS03 | Po1f cells harboring p1312HMG1 and p1269ERG20 | This study |
| CXS04 | Po1f cells harboring p1312HMG1 and p1269ERG9 | This study |
| CXS05 | Po1f cells harboring p1312HMG1 and p1269ERG9ERG10 | This study |
| CXS06 | Po1f cells harboring p1312HMG1 and p1269ERG9ERG12 | This study |
| CXS07 | Po1f cells harboring p1312HMG1 and p1269ERG9ERG20 | This study |
| CXS08 | Po1f cells harboring p1312HMG1 and p1269DGA1 | This study |
| CXS09 | Po1f cells harboring p1312HMG1 and p1269ERG9 DGA1 | This study |
| CXS10 | Po1f in which PEX10 has been deleted | This study |
| CXS11 | Po1f in which PEX10 has been deleted and URA3 has been recycled | This study |
| CXS12 | Po1f in which PEX10 has been deleted and harboring p1312HMG1 | This study |
| CXS13 | Po1f in which URE2 has been deleted | This study |
| CXS14 | Po1f in which URE2 has been deleted and URA3 has been recycled | This study |
| CXS15 | Po1f in which URE2 has been deleted and harboring p1312HMG1 | This study |
| CXS16 | Po1f in which PEX10 and URE2 have been deleted | This study |
| CXS17 | Po1f in which PEX10 and URE2 have been deleted and URA3 has been recycled | This study |
| CXS18 | Po1f in which PEX10 and URE2 have been deleted and harboring p1312HMG1 | This study |
| CXS19 | Po1f in which PEX10 and URE2 have been deleted and harboring p1312HMG1 and p1269DGA1 | This study |
| CXS20 | Po1f in which PEX10 and URE2 have been deleted and harboring p1312HMG1 and p1269DGA1ERG9 | This study |
Plasmid construction.
Standard procedures or manufacturers’ instructions were followed for plasmid construction. The genes HMG1, ERG10, ERG12, ERG20, ERG9, and DGA1, amplified from genomic DNA of Po1f, were cloned into pINA1312 or pINA1269 with primers P1/P2, P7/P8, P9/P10, P13/P14, P11/P12, and P15/P16. This formed plasmids p1312HMG1, p1269ERG10, p1269ERG12, p1269ERG20, p1269ERG9, and p1269DGA1, respectively. The expression cassettes P-ERG10-T, P-ERG12-T, P-ERG20-T, and P-DGA1-T then were cloned into p1269ERG9 with primers P17/P18 to obtain p1269ERG9ERG10, p1269ERG9ERG12, p1269ERG9ERG20, and p1269ERG9DGA1, respectively.
Inactivation of the PEX10 and URE2 genes was performed using a previously reported gene disruption cassette for repeated use in Y. lipolytica (68). Gene disruption cassettes containing the URA3 selectable marker flanked by loxP sites (obtained by PCR of the JMP113 plasmid) were produced with 1 to 1.5 kb of homology on either side of each target integration site. All of the plasmids were constructed using the One Step cloning kit from Vazyme Biotech Co., Ltd. (Nanjing, China) and are listed in Table 1. Oligonucleotide primers used for PCR, cloning, and deletion in this study are included Table S1 in the supplemental material.
Strain construction.
The integrative fragments or linear plasmids were transformed in Y. lipolytica. For gene deletion, the marker gene (URA3) was removed by overexpressing the Cre recombinase in the pUB-Cre plasmid. To excise the selection marker between the loxP sites in the disruption cassette, detailed procedures were followed according to the protocols in previously published work (68). This enables subsequent rounds of genomic integrations. All the transformations were performed with Zymogen frozen EZ yeast transformation II (Zymo Research Corporation), and the colonies were selected on minimal medium lacking uracil or leucine. The resulting strains were confirmed through genomic DNA extraction and PCR confirmation and listed in Table S1.
Squalene and ergosterol analysis.
Quantification of squalene and ergosterol in Y. lipolytica was performed as previously described (17). Cultured cells were collected and then resuspended in an alcoholic potassium hydroxide solution (20% [wt/vol] in 50% ethanol) and vortexed on high for 20 min. After boiling for 5 min, the samples were immediately cooled on ice. Next, the samples were extracted with 600 μl hexane before being dried with a vacuum dryer. The dried samples were suspended in 50 μl ethanol and 450 μl acetonitrile, and 10-μl samples were analyzed by a Shimadzu LC-20AD high-performance liquid chromatography system equipped with a SinoChrom ODS-BP column (4.6 by 250 mm, 5 μm; Dalian Elite Analytical Instruments Co., Ltd.), and UV detection was performed at 195 nm. The samples were eluted with 100% acetonitrile at a flow rate of 2 ml/min at 35°C. The peak areas were proportional to the concentration. All results were reported as averages from three replicates.
Lipid quantification and fatty acid profile analysis.
Lipids from a 30-ml volume of fermentation broth were extracted after 96 h by following the procedure described by Nambou et al. (69). Briefly, Y. lipolytica cells were spun down and washed with water once and resuspended in an HCl solution (60:80 [vol/vol]) and heated to boiling. After cooling on ice, a chloroform-methanol solution (3:2) was added, and the whole mixture was incubated at 37°C in a shaker incubator for almost 1 h. The organic solution was extracted before being purified by distillation under vacuum at 80°C, dried at 105°C for 1 h, and weighed to quantify lipid production. The dried lipids were transesterified with a solution of boron trifluoride methanol by following a previous procedure (69), and 0.2-μl samples were injected into an Agilent 6890-5975 (gas chromatography-mass spectrometry [GC-MS]) (Agilent Technologies, Santa Clara, CA). The HP-5 MS column (30 m by 0.25 mm by 0.25 μm) (Agilent Technologies) was used for GC-MS analysis. GC oven temperature was programmed from 180°C (2 min) to 250°C at 5°C/min, and the flow rate of carrier gas was set at 1 ml/min. Other settings were 250°C interface temperature, 230°C ion source temperature, and electron impact ionization (EI) at −70 (or 70) eV, with a full scan ranging from 70 to 560 m/z and a solvent delay of 1.5 min.
Transcriptional analysis by quantitative real-time RT-PCR.
The cells were pelleted and total RNA was extracted using TRIzol reagent (Sigma-Aldrich, St. Louis, MO) according to protocol and treated with DNase to remove the residual genomic DNA. RNA samples were used for a reverse transcription reaction with the FastQuant RT kit (with gDNase) (Tiangen Biotech Co., Beijing, China). Subsequently, quantitative PCRs (qRT-PCRs) were performed with SuperReal premix plus (SYBR green) (Tiangen Biotech). All reactions were done in triplicate and performed in a Bio-Rad CFX Fast real-time PCR system (Hercules, CA). To prevent variation of the starting materials amounts, actin was conserved and used as a loading control. The results were normalized to actin gene expression and then analyzed according to the ΔΔCT method (70). All the primers used for quantitative PCR are shown in Table S2.
Supplemental Material
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Information & Contributors
Information
Published In
Applied and Environmental Microbiology
Volume 87 • Number 17 • 11 August 2021
eLocator: e00481-21
Editor: Haruyuki Atomi, Kyoto University
PubMed: 34132586
Copyright
© 2021 American Society for Microbiology. All Rights Reserved.
History
Received: 10 March 2021
Accepted: 29 May 2021
Accepted manuscript posted online: 16 June 2021
Published online: 11 August 2021
Keywords
Contributors
Editor
Haruyuki Atomi
Editor
Kyoto University
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2021.
Increased Accumulation of Squalene in Engineered Yarrowia lipolytica through Deletion of PEX10 and URE2. Appl Environ Microbiol
87:e00481-21.
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