Metabolic recycling of storage lipids promotes squalene biosynthesis in yeast
Biotechnology for Biofuels and Bioproducts volume 15, Article number: 108 (2022)
Metabolic rewiring in microbes is an economical and sustainable strategy for synthesizing valuable natural terpenes. Terpenes are the largest class of nature-derived specialized metabolites, and many have valuable pharmaceutical or biological activity. Squalene, a medicinal terpene, is used as a vaccine adjuvant to improve the efficacy of vaccines, including pandemic coronavirus disease 2019 (COVID-19) vaccines, and plays diverse biological roles as an antioxidant and anticancer agent. However, metabolic rewiring interferes with inherent metabolic pathways, often in a way that impairs the cellular growth and fitness of the microbial host. In particular, as the key starting molecule for producing various compounds including squalene, acetyl-CoA is involved in numerous biological processes with tight regulation to maintain metabolic homeostasis, which limits redirection of metabolic fluxes toward desired products.
In this study, focusing on the recycling of surplus metabolic energy stored in lipid droplets, we show that the metabolic recycling of the surplus energy to acetyl-CoA can increase squalene production in yeast, concomitant with minimizing the metabolic interferences in inherent pathways. Moreover, by integrating multiple copies of the rate-limiting enzyme and implementing N-degron-dependent protein degradation to downregulate the competing pathway, we systematically rewired the metabolic flux toward squalene, enabling remarkable squalene production (1024.88 mg/L in a shake flask). Ultimately, further optimization of the fed-batch fermentation process enabled remarkable squalene production of 6.53 g/L.
Our demonstration of squalene production via engineered yeast suggests that plant- or animal-based supplies of medicinal squalene can potentially be complemented or replaced by industrial fermentation. This approach will also provide a universal strategy for the more stable and sustainable production of high-value terpenes.
Metabolic rewiring in microbes has been proposed as a promising strategy for the economical and sustainable production of valuable medicinal compounds that are found in trace quantities in nature [1,2,3,4]. Metabolic pathways have been rewired to unlock metabolic bottlenecks by enhancing the supply of metabolic building blocks or reducing metabolic flux-competing reactions toward the desired compounds . However, metabolic rewiring interferes with inherent metabolic pathways, often in a way that impairs the cellular growth and fitness of the microbial host. In particular, as the key starting molecule for producing various compounds, acetyl-CoA is involved in numerous biological processes with tight regulation to maintain metabolic homeostasis, which limits redirection of metabolic fluxes toward desired products [1, 6].
Lipid droplets (LDs) are energy storehouses of surplus metabolic energy and lipids, supplying the cells with metabolic building blocks such as acetyl-CoA for energy generation when the cells are starved [7, 8]. This implies that the breakdown of surplus lipids in LDs can provide sufficient acetyl-CoA for cellular growth and overall fitness of the host cells, allowing the concomitant overproduction of the desired target compounds. We thus explored the metabolic recycling of acetyl-CoA from the surplus energy in LDs for the production of medicinal terpenes while minimizing the metabolic interferences in the inherent pathways. Here, we performed systematic metabolic rewiring of Saccharomyces cerevisiae toward squalene production (Fig. 1). As a model medicinal terpenes, squalene is a high-value natural compound that has been shown to play diverse biological roles as an antioxidant and anticancer agent and has been employed as a vaccine adjuvant to improve the efficacy of vaccines, including pandemic coronavirus disease 2019 (COVID-19) vaccines [9, 10]. Although increased consumer demand has prompted the development of microbial bioprocesses for squalene production, these cannot meet the growing demand for squalene.
In this study, we first controlled the intrinsic mevalonate (MVA) pathway by multiple integration of the rate-limiting enzyme tHmg1 (a cytosolic non-feedback-inhibited 3-hydroxy-3-methylglutaryl-CoA reductase) into the genome of our platform yeast strain for terpene production (SQ00) . Subsequently, driven by an N-degron-dependent protein degradation strategy [12, 13], we downregulated Erg1 (a squalene epoxidase catalyzing the oxidation of squalene to 2,3-oxidosqualene) activity. We further rewired LD metabolism to improve the acetyl-CoA supply, which resulted in significantly increased production of squalene (1024.88 mg/L by batch fermentation in a shake flask). Ultimately, further optimization of the fed-batch fermentation process enabled remarkable squalene production of 6.53 g/L. Our study offers an efficient strategy to enhance squalene production and might also be applicable for the production of other valuable compounds.
Results and discussion
Construction of a squalene-overproducing pathway in S. cerevisiae
Squalene is naturally synthesized through the MVA pathway, involving multiple enzymes, in yeast (Fig. 1). Metabolic fluxes through the MVA pathway are under tight feedback regulation from pathway intermediates or downstream products [14, 15]; for instance, HMG, free CoA, and NAD(P)+/NADPH inhibiting HMG–CoA reductase (Hmg1)  and sterol inhibiting squalene epoxidase (Erg1)  catalyze the rate-limiting step, thereby controlling the squalene biosynthesis flux. Therefore, to construct a strain for high-level production of squalene, we investigated the effects of the rate-limiting enzymes in squalene biosynthesis.
First, we overexpressed a truncated HMG1 (tHMG1) gene encoding the catalytic domain of Hmg1, which has been reported as the major rate-limiting enzyme to enhance metabolic fluxes in the MVA pathway and squalene production , in a yeast biosynthetic platform for terpene production developed by our group, the SQ00 strain  (Additional file 1: Table S1). The strain SQ00, designed to increase terpene production, incorporated (i) overexpression of FPP synthetase (Erg20), which supplies sufficient FPP for terpene synthesis [11, 18], and (ii) expansion of the endoplasmic reticulum (ER) by overexpressing a key ER size regulatory factor (Ino2) for functional assembly of the terpene synthesis pathway . We integrated the tHMG1 gene into a multicopy δ-sequence of the SQ00 genome (Fig. 1), generating a series of squalene-producing strains in which the yields of squalene production varied from 11.10 mg/L to 550.89 mg/L after 144 h of fermentation (Fig. 2A; Additional file 1: Fig. S1). The δ-integration strategy has been widely used for the overexpression of heterologous genes to construct biosynthesis pathways, as it can simultaneously achieve random multicopy integration of target genes [19, 20].
The copy numbers of the integrated tHMG1 gene were investigated using quantitative PCR analysis (Fig. 2A). The transformants with squalene production titers over 500 mg/L contained 4 copies of the tHMG1 gene. Furthermore, we found that as the copy numbers of the tHMG1 gene increased, its transcription level gradually increased up to 4 copies, but rather decreased when the copy numbers are further increased (Fig. 2B). This observation indicates that the copy numbers of the integrated tHMG1 gene is indeed positively correlated with its expression as well as squalene production titer up to a certain copy number level. Thus, among those strains, one strain with the highest squalene titer (550.89 mg/L) and the tHMG1 expression level (4 copies) as well was selected and referred to as the SQ4t strain, which had an almost 133-fold greater squalene titer than the control strain SQ00 (4.14 mg/L). The variations in squalene production among strains with the same copy number of tHMG1 were speculated to be affected by different δ-integration sites, because it was recently shown that the position effect accounts for increased variability in gene expression levels .
Second, to further improve squalene production by the SQ4t strain, we modulated the ergosterol biosynthesis pathway, which converts squalene to ergosterol (Fig. 1). Ergosterol is an essential membrane component, with tightly controlled homeostasis in yeast. Accordingly, as most genes required for ergosterol synthesis are vital for cell growth, they cannot be deleted. Transcriptional downregulation of ERG1, the first enzyme catalyzing the epoxidation of squalene to 2,3-oxidosqualene in the ergosterol biosynthesis pathway, also leads to growth defects, causing failure to engineer yeast suitable for industrial-scale production of squalene [12, 13]. Although partial inhibition of Erg1 activity with the addition of its inhibitor terbinafine was effective for squalene accumulation, as reported previously , it showed a pronounced reduction in cell growth and squalene titer with increasing terbinafine concentration (Fig. 2C; Additional file 1: Table S3). In addition, terbinafine is a highly lipophilic supplement that is difficult to remove through the downstream process of purification for squalene production.
Therefore, we utilized the N-degron-mediated protein degradation strategy to reduce Erg1 activity concomitant with the maintenance of ergosterol flux for normal cell growth and coupled it with an ergosterol-responsive transcription modulator circuit (Fig. 1) . The N-degron tag is a ubiquitin moiety and a destabilizing sequence enriched with lysine and asparagine, which are sequentially fused to the N-terminus of a target protein [23, 24]. The resulting fusion protein is hydrolyzed by ubiquitin C-terminal hydrolase, and the destabilizing sequence is exposed following N-rule protein degradation. Therefore, the N-degron tag can result in a reduced half-life or deletion-like phenotype of the target protein by inducing proteasomal proteolysis. In this study, to reduce metabolic flux toward ergosterol synthesis, we modified the genomic ERG1 gene by fusing the N-degron to the Erg1p N-terminus (Additional file 1: Table S2). Furthermore, as noted, the ERG1 gene under the control of an ergosterol-responsive promoter is regulated inversely proportionally to ergosterol concentration . Consequently, degron-tagged ERG1 expression was paired with the ergosterol-responsive promoter, thus increasing squalene accumulation while ensuring sufficient metabolic flux to essential downstream ergosterol synthesis.
The resulting strain expressing the N-degron-tagged Erg1 in the SQ4t strain (SQ4td strain) produced 720.47 mg/L squalene after 144 h of fermentation, and this value exhibited an ~ 31% increase over that produced by the control SQ4t strain (550.89 mg/L), without a reduction in cell growth (Fig. 2C; Additional file 1: Table S3). This result indicates that with the aim of minimizing carbon flux toward ergosterol synthesis, the N-degron-mediated degradation of Erg1 successfully reduces carbon flux toward ergosterol synthesis and redirects the carbon flux into squalene production, similar to the effect of using terbinafine but without compromising cell growth.
Redirection of LD metabolism to boost squalene production
Neutral lipids stored in LDs are mobilized in the form of free fatty acids (FFAs) for energy production during nutrient deprivation or for phospholipid synthesis during the high demand of membrane production [26, 27]. FFAs released from LDs through lipolysis can be redirected toward the supply of acetyl-CoA, a key metabolic building block for many biochemical reactions. Previous studies reported that hydrolyzing TAG to fatty acids (FAs) and diacylglycerol (DAG) may influence acetyl-CoA availability via subsequent metabolism through β-oxidation (Fig. 1) [28,29,30]. For this reason, we hypothesized that recycling of FAs stored in LDs can support an increase in the acetyl-CoA supply, favoring squalene production.
To verify this hypothesis, we overexpressed four different genes involved in TAG lipolysis or acyl-CoA synthesis in the SQ4td strain. In detail, we overexpressed the TGL3 or TGL4 gene encoding the main TAG-lipase involved in FA supply , generating SQ4td-TGL3 and SQ4td-TGL4. The FAA1 or FAA4 gene (encoding a acyl-CoA synthetase), which produces acyl-CoA from FAs , was overexpressed in SQ4td to generate SQ4td-FAA1 or SQ4td-FAA4. The level of squalene production was the highest (862.62 mg/L) in the SQ4td-TGL3 strain after 144 h of cultivation, which was approximately 20% higher than that by SQ4td (720.47 mg/L) (Fig. 3). In addition, the SQ4td-TGL4 and SQ4td-FAA1 strains produced higher levels of squalene (816.48 and 774.07 mg/L, respectively) than SQ4td. However, squalene production in the SQ4td-FAA4 strain was not increased (723.90 mg/L), which is consistent with a previous report demonstrating that Faa1 rather than Faa4 acts as the predominant fatty acyl-CoA synthetase . In addition, the growth of LD metabolism-engineered strains was similar to that of SQ4td, suggesting that the overexpression of the four genes for recycling LD-dependent FAs did not inhibit cell growth (Additional file 1: Table S4). In an attempt to further increase squalene production, we combined overexpression of the TGL3 and TGL4 genes (SQ4td-TGL3/4 strain), but no obvious improvement in squalene production was observed (832.63 mg/L) (Additional file 1: Table S4).
Thus, we performed additional analyses for only single mutants except for the SQ4td-TGL3/4 strain. First, we evaluated whether acetyl-CoA content was increased by rewiring LD metabolism. The level of acetyl-CoA in SQ4td-TGL3 was highly increased by ~ 24% (0.84 pmol/g dry cell weight (DCW)) at 72 h compared with that in the SQ4td strain (0.68 pmol/g DCW) (Fig. 4A). Moreover, we observed that the highest level of acetyl-CoA was sustained in the SQ4td-TGL3 strain during cultivation for 144 h, which was consistent with the finding that the SQ4td-TGL3 strain produced squalene most productively. This result might be due to an increase in acetyl-CoA supply by TAG mobilization or degradation via a cascade of hydrolysis reactions from TAG and DAG to FAs.
Next, to further clarify the effect of acetyl-CoA supply on TAG degradation, TAG levels were quantified in the engineered strains. The level of TAG was decreased by ~ 47% (3.47 nmol/g DCW) in the highest squalene-overproducing strain SQ4td-TGL3 compared with that in the SQ4td strain (6.59 nmol/g DCW) at 72 h (Fig. 4B). The effect of TAG lipolysis was maintained from 24 to 72 h of cultivation in the SQ4td-TGL3 strain. Consistent with the decreased TAG level, the SQ4td-TGL3 strain had significantly fewer LDs than the SQ4td strain; the number of LDs in the SQ4td-TGL3 strain decreased by ~ 39% (7.8 LDs per cell), with no significant difference in the average diameter of the LDs (Davg ~ 0.58 µm), resulting in an ~ 43% decrease in the total LD volume per cell (0.91 µm3) compared to those in the SQ4td strain (12.7 LDs per cell, average diameter of 0.58 µm and volume per cell of 1.60 µm3) (Fig. 4C, D; Additional file 1: Fig. S2). Consequently, LD metabolism rewiring by TGL3 overexpression increased the acetyl-CoA supply, which could explain its larger contribution to squalene overproduction.
Optimization of culture conditions for enhanced squalene production
For the engineered yeast strain with the most promising performance for squalene production, the choice of carbon source was important for the production efficiency. Ethanol is often used as the main carbon source in fed-batch fermentation due to increased titers in the production of many terpenes . Glycerol is another potential source for yeast due to its higher reduced state of carbon compared to that of other sugars . However, glucose is still a widely used carbon source in industrial terpene production.
Therefore, we tested three different carbon sources to determine which would be optimal for the production of squalene. The best strain SQ4td-TGL3 was cultivated in shake flasks with glucose, glycerol, or ethanol (2%) as the carbon source, and the highest titer of 862.62 mg/L squalene was produced from glucose as a sole carbon source after 144 h of cultivation (Fig. 5). In contrast, we observed significantly lower titers of squalene (115.90 and 279.75 mg/L after 144 h of cultivation) from glycerol and ethanol, respectively.
Previously, studies have shown that when mixing carbon sources, cells tend to coutilize the carbon sources, and the growth rate or the yield of target products is higher than that with each individual source . Thus, we further investigated squalene production using mixed carbon sources, namely, glucose/glycerol, glycerol/ethanol, and glucose/ethanol mixed carbon sources (1:1, 1% v/v each) (Fig. 5). However, when cultivated on a mixture of two carbon sources, the strain SQ4td-TGL3 produced lower levels of squalene: 505.37 mg/L on glucose/glycerol, 308.53 mg/L glycerol/ethanol, and 695.61 mg/L glucose/ethanol. This result indicates that glycerol and ethanol were not suitable as the carbon source in our strain and that utilization of these nonfermentable carbon sources might be inhibited in the presence of glucose [36, 37].
In general, yeast cells grow more vigorously in complex media than in defined media; since complex media include unknown ingredients, they are rich in nutrients and other necessary elements to support cellular growth [38, 39]. Notably, when cultivated in the complex medium with glucose, the strain SQ4td-TGL3 showed a 26% higher squalene titer (1024.88 mg/L) after 144 h of cultivation compared to that in the defined medium (Fig. 5). Finally, in a 5-L bioreactor, we realized greater improvements in the final production titer, which reached 6.53 g/L squalene in the fed-batch mode using the complex medium with glucose as the carbon source (Fig. 6).
Squalene is a crucial intermediate and precursor for valuable bioactive compounds used in the food, drug, chemical and cosmetic industries. Given the growing demand for squalene, three to six million deep sea sharks are slaughtered each year. A global health crisis, such as the ongoing COVID-19 pandemic, has resulted in the further increased need for squalene, which is an ingredient currently used in influenza vaccines. To overcome this unsustainable practice, we engineered baker’s yeast to produce the high-value chemical squalene, starting from simple sugars. This was accomplished by (i) overexpressing the native rate-limiting enzyme encoded by the tHMG1 gene, (ii) controlling the competitive ergosterol biosynthesis pathway using the N-degron-dependent protein degradation strategy, (iii) improving acetyl-CoA supply via the metabolic recycling of the surplus energy of LDs, and (iv) optimizing the fermentation medium, including the carbon source and the cultivation medium. Overall, we systematically rewired the metabolic flux toward squalene, enabling remarkable squalene production (1024.88 mg/L in the shake flask and 6.53 g/L in the fed-batch fermenter). Our demonstration of squalene production via engineered yeast suggests that plant- or animal-based supplies of medicinal squalene can potentially be complemented or replaced by industrial fermentation.
Plasmid and strain construction
All plasmids, strains and primers are listed in Additional file 1: Tables S1 and S2. The plasmids used in this study were generated via insertion of the gene fragment, which was amplified from the yeast genomic DNA with corresponding primer pairs and digested with restriction enzymes, into pUC57-URA3-derived vectors for strain construction. Gene modifications were introduced by the URA3-blaster genetic disruption method . Recombination cassettes for gene integration or promoter replacement were amplified by PCR from pUC57-URA3-derived vectors containing a gene of interest or the appropriate promoter, respectively, with primer pairs introducing regions homologous to the target recombination site. The yeast plasmids were transformed into the recombinant strains by the standard LiAc/ssDNA/PEG method .
Shake flask fermentation for squalene production
A total of 50 mL of yeast synthetic complete (YSC) medium (0.19% yeast synthetic dropout medium without uracil and 0.67% yeast nitrogen base without amino acids) supplemented with 2% (w/v) glucose was used to culture engineered yeast strains for the production of squalene. First, engineered strains were grown on YSC agar plates with 2% (w/v) glucose lacking uracil. A yeast colony was inoculated into 50 mL conical tubes with 10 mL of YSC seed medium. After overnight cultivation at 30 °C with shaking at 250 rpm, the seed cultures were inoculated into 250 mL flasks with 50 mL of YSC medium containing a 2% carbon source to reach an initial optical density (OD600) of 0.5 and then cultivated for 6 days at 30 °C with shaking at 250 rpm. For the carbon sources in each medium, the following concentrations were applied: 2% (w/v) glucose, 2% (v/v) glycerol, and 2% (v/v) ethanol. All flask fermentations were performed in three independent experiments.
Determination of gene copy number and gene expression level
To determine the copy numbers of the tHMG1 gene, quantitative PCR (qPCR) was performed with tHMG1-specific primers and iTaq Universal SYBR Green Supermix (Bio-Rad, CA, USA). qPCR was performed on a CFX96™ Real-Time PCR Detection System (Bio-Rad, CA, USA) with the following conditions: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 15 s [42, 43]. The TAF10 gene was used as a reference control. The crossing point (Cp) values were determined using Bio-Rad CFX Manager software (Bio-Rad, CA, USA).
For analyzing gene expression level of the tHMG1 gene, RNA was extracted from the cells using RNeasy kit (Qiagen, Hilden, Germany) at the time intervals as indicated. Then, the cDNA was synthesized with a Superscript III First-Strand Synthesis System (Life Technologies, OR, USA) according to the manufacturer’s instructions. The relative gene expression level was quantified by the comparative threshold cycle (2−∆∆Ct) method . The primers used are listed in Additional file 1: Table S1. All experiments were performed in triplicate.
Fed-batch fermentation for squalene production
Fed-batch fermentations were performed in a 5 L fermenter (CNS, Daejeon, Korea). The initial working volume was 2 L of YP medium (10 g/L yeast extract and 20 g/L peptone) supplemented with 2% (w/v) glucose and 200 mL of overnight preculture in the same medium. Fed-batch fermentation was carried out at 30 °C with an agitation speed of 600 rpm and an airflow rate of 1 v.v.m. The pH was maintained at 6.0 by automatic feeding of a 2 M NaOH solution. The temperature, pH, agitation, and dissolved oxygen concentration were monitored and controlled using a CNS control system. A feeding solution comprising 600 g/L glucose and 40 g/L yeast extract was added at a feeding rate of < 5 g/L/h after the glucose was completely consumed.
Metabolite extraction and analysis
To extract squalene, yeast cells were harvested by centrifugation at 13,000×g for 5 min to obtain an equivalent OD600 of 10. The harvested cells were resuspended in 600 µL of a 1:1 methanol–acetone (MA) solution with lysing matrix C. Subsequently, the mixture was mechanically disrupted using a FastPrep-24 5G homogenizer (MP Biomedicals, CA, USA) according to the manufacturer’s instructions. After filtration using 0.2-μm syringe filters, squalene extracted from the MA solution was analyzed using an Agilent high-performance liquid chromatography (HPLC) system equipped with a UV detector at 203 nm. Squalene was separated on a Kinetex 5 µm EVO C18 column (Phenomenex, Aschaffenburg, Germany) at 30 °C with an isocratic elution flow rate of 1.0 mL/min for 30 min. The mobile phase comprised acetonitrile, methanol, and water at a ratio of 90:9:1 (v/v).
Glucose, glycerol, ethanol and organic acid concentrations were determined with an HPLC system equipped with an Aminex HPX-87G column (Bio-Rad, CA, USA). In detail, a 1 mL culture sample was centrifuged and filtered through a 0.2-µm syringe filter and then analyzed on the HPLC system with 5 mM H2SO4 as the mobile phase at a flow rate of 0.6 mL/min at 45 °C for 30 min.
Quantification of acetyl-CoA and TAG
Acetyl-CoA and TAG were quantified as described previously, with minor modifications [45,46,47,48]. Briefly, yeast cells were grown in YSC medium overnight at 30 °C with shaking at 250 rpm. The precultures were then inoculated into 50 mL of YSC medium containing 2% (w/v) glucose at an initial OD600 of 0.5 and cultivated for 6 days at 30 °C with shaking at 250 rpm. At appropriate timepoints, yeast cells were harvested by centrifugation at 13,000 rpm for 5 min at 4 °C.
For quantification of acetyl-CoA, 10 OD600 of cells were resuspended in 500 μL of a cold Tris-EDTA (pH 8.0) buffer solution (Sigma-Aldrich, Missouri, USA). Extracts were prepared with a FastPrep-24 5G homogenizer (MP Biomedicals, OH, USA) using lysing matrix C. After centrifuging at 13,000 rpm for 5 min at 4 °C, the supernatant was used for acetyl-CoA measurement with a PicoProbe™ Acetyl-CoA Fluorometric Assay Kit (Biovision, Milpitas, USA) according to the manufacturer's instructions. Acetyl-CoA concentrations were measured by fluorescence intensity at wavelengths 535 nm (excitation) and 587 nm (emission) on a microplate reader (SpectraMax Gemini XPS, Molecular Devices, CA, USA). For quantification of TAG, 2 OD600 of cells were homogenized in 1 mL of 5% NP-40 (Sigma-Aldrich, Missouri, USA). Using the homogenates, TAG was quantified using a Triglyceride Quantification Kit (BioVision, Milpitas, USA) according to the manufacturer's instructions.
Confocal fluorescence microscopy
To analyze the size and number of LDs, yeast cells were grown in YSC medium supplemented with 2% (w/v) glucose. After growing for 24 h at 30 °C with shaking at 250 rpm, the cells were washed twice with phosphate-buffered saline (PBS) and fixed with 3.7% formaldehyde in PBS as described previously [11, 49, 50]. Next, the cells were stained with 5 µM BODIPY 493/503 dye for 30 min at 30 °C. Then, the cells were washed twice with PBS and observed with a Zeiss-LSM 780 multiphoton confocal microscope (Zeiss, AG, Germany) equipped with a Plan-Apochromat 63x/1.4 NA oil immersion objective. Confocal images were analyzed using ImageJ software (National Institutes of Health, Bethesda, USA) and ZEN imaging software (Zeiss, AG, Germany).
Figures were prepared using BioRender.Com for scientific illustrations.
Nielsen J, Keasling JD. Engineering cellular metabolism. Cell. 2016;164(6):1185–97.
Romero-Suarez D, Keasling JD, Jensen MK. Supplying plant natural products by yeast cell factories. Curr Opin Green Sustain Chem. 2022;33: 100567.
Rahmat E, Kang Y. Yeast metabolic engineering for the production of pharmaceutically important secondary metabolites. Appl Microbiol Biotechnol. 2020;104(11):4659–74.
Lee SK, Chou H, Ham TS, Lee TS, Keasling JD. Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels. Curr Opin Biotechnol. 2008;19(6):556–63.
Lian J, Mishra S, Zhao H. Recent advances in metabolic engineering of Saccharomyces cerevisiae: new tools and their applications. Metab Eng. 2018;50:85–108.
Krivoruchko A, Zhang Y, Siewers V, Chen Y, Nielsen J. Microbial acetyl-CoA metabolism and metabolic engineering. Metab Eng. 2015;28:28–42.
Olzmann JA, Carvalho P. Dynamics and functions of lipid droplets. Nat Rev Mol Cell Biol. 2019;20(3):137–55.
Welte MA, Gould AP. Lipid droplet functions beyond energy storage. Biochim Biophys Acta Mol Cell Biol Lipids. 2017;1862(10 Pt B):1260–72.
Kim EH, Woodruff MC, Grigoryan L, Maier B, Lee SH, Mandal P, Cortese M, Natrajan MS, Ravindran R, Ma H, et al. Squalene emulsion-based vaccine adjuvants stimulate CD8 T cell, but not antibody responses, through a RIPK3-dependent pathway. Elife. 2020, 9.
Macdonald C, Soll J. Shark conservation risks associated with the use of shark liver oil in SARS-CoV-2 vaccine development. bioRxiv. 2020;110:267.
Kim J-E, Jang I-S, Son S-H, Ko Y-J, Cho B-K, Kim SC, Lee JY. Tailoring the Saccharomyces cerevisiae endoplasmic reticulum for functional assembly of terpene synthesis pathway. Metab Eng. 2019;56:50–9.
Rasool A, Ahmed MS, Li C. Overproduction of squalene synergistically downregulates ethanol production in Saccharomyces cerevisiae. Chem Eng Sci. 2016;152:370–80.
Garaiová M, Zambojová V, Šimová Z, Griač P, Hapala I. Squalene epoxidase as a target for manipulation of squalene levels in the yeast Saccharomyces cerevisiae. FEMS Yeast Res. 2014;14(2):310–23.
Wei LJ, Kwak S, Liu JJ, Lane S, Hua Q, Kweon DH, Jin YS. Improved squalene production through increasing lipid contents in Saccharomyces cerevisiae. Biotechnol Bioeng. 2018;115(7):1793–800.
Siddiqui MS, Thodey K, Trenchard I, Smolke CD. Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools. FEMS Yeast Res. 2012;12(2):144–70.
Partow S, Siewers V, Daviet L, Schalk M, Nielsen J. Reconstruction and evaluation of the synthetic bacterial MEP pathway in Saccharomyces cerevisiae. PLoS ONE. 2012;7(12): e52498.
Sun L, Atkinson CA, Lee YG, Jin YS. High-level β-carotene production from xylose by engineered Saccharomyces cerevisiae without overexpression of a truncated HMG1 (tHMG1). Biotechnol Bioeng. 2020;117(11):3522–32.
Li T, Liu GS, Zhou W, Jiang M, Ren YH, Tao XY, Liu M, Zhao M, Wang FQ, Gao B, et al. Metabolic engineering of Saccharomyces cerevisiae to overproduce squalene. J Agric Food Chem. 2020;68(7):2132–8.
Yuan J, Ching CB. Combinatorial assembly of large biochemical pathways into yeast chromosomes for improved production of value-added compounds. ACS Synth Biol. 2015;4(1):23–31.
Yamada R, Taniguchi N, Tanaka T, Ogino C, Fukuda H, Kondo A. Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains. Microb Cell Fact. 2010;9(1):1–8.
Chen M, Licon K, Otsuka R, Pillus L, Ideker T. Decoupling epigenetic and genetic effects through systematic analysis of gene position. Cell Rep. 2013;3(1):128–37.
Han JY, Seo SH, Song JM, Lee H, Choi E-S. High-level recombinant production of squalene using selected Saccharomyces cerevisiae strains. J Ind Microbiol Biotechnol. 2018;45(4):239–51.
Peng B, Nielsen LK, Kampranis SC, Vickers CE. Engineered protein degradation of farnesyl pyrophosphate synthase is an effective regulatory mechanism to increase monoterpene production in Saccharomyces cerevisiae. Metab Eng. 2018;47:83–93.
Nguyen KT, Mun S-H, Lee C-S, Hwang C-S. Control of protein degradation by N-terminal acetylation and the N-end rule pathway. Exp Mol Med. 2018;50(7):1–8.
Jordá T, Puig S. Regulation of ergosterol biosynthesis in Saccharomyces cerevisiae. Genes. 2020;11(7):795.
Cabodevilla AG, Sánchez-Caballero L, Nintou E, Boiadjieva VG, Picatoste F, Gubern A, Claro E. Cell survival during complete nutrient deprivation depends on lipid droplet-fueled β-oxidation of fatty acids. J Biol Chem. 2013;288(39):27777–88.
Xu C, Fan J. Links between autophagy and lipid droplet dynamics. J Exp Bot. 2022;73:2848.
Chen Y, Daviet L, Schalk M, Siewers V, Nielsen J. Establishing a platform cell factory through engineering of yeast acetyl-CoA metabolism. Metab Eng. 2013;15:48–54.
Lian J, Si T, Nair NU, Zhao H. Design and construction of acetyl-CoA overproducing Saccharomyces cerevisiae strains. Metab Eng. 2014;24:139–49.
Klug L, Daum G. Yeast lipid metabolism at a glance. FEMS Yeast Res. 2014;14(3):369–88.
Kurat CF, Wolinski H, Petschnigg J, Kaluarachchi S, Andrews B, Natter K, Kohlwein SD. Cdk1/Cdc28-dependent activation of the major triacylglycerol lipase Tgl4 in yeast links lipolysis to cell-cycle progression. Mol Cell. 2009;33(1):53–63.
Færgeman NJ, Black PN, Zhao XD, Knudsen J, DiRusso CC. The Acyl-CoA synthetases encoded within FAA1 andFAA4 in Saccharomyces cerevisiae function as components of the fatty acid transport system linking import, activation, and intracellular utilization. J Biol Chem. 2001;276(40):37051–9.
Zhao J, Li C, Zhang Y, Shen Y, Hou J, Bao X. Dynamic control of ERG20 expression combined with minimized endogenous downstream metabolism contributes to the improvement of geraniol production in Saccharomyces cerevisiae. Microb Cell Fact. 2017;16(1):1–11.
Xiberras J, Klein M, Nevoigt E. Glycerol as a substrate for Saccharomyces cerevisiae based bioprocesses—knowledge gaps regarding the central carbon catabolism of this ‘non-fermentable’carbon source. Biotechnol Adv. 2019;37(6): 107378.
Wang X, Xia K, Yang X, Tang C. Growth strategy of microbes on mixed carbon sources. Nat Commun. 2019;10(1):1279.
Kayikci Ö, Nielsen J. Glucose repression in Saccharomyces cerevisiae. FEMS Yeast Res. 2015;15(6):fov068.
Santangelo GM. Glucose signaling in Saccharomyces cerevisiae. Microbiol Mol Biol Rev. 2006;70(1):253–82.
La Grange DC, Pretorius IS, Van Zyl WH. Expression of a Trichoderma reesei beta-xylanase gene (XYN2) in Saccharomyces cerevisiae. Appl Environ Microbiol. 1996;62(3):1036–44.
Choi E-S, Sohn J-H, Rhee S-K. Optimization of the expression system using galactose-inducible promoter for the production of anticoagulant hirudin in Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 1994;42(4):587–94.
Alani E, Cao L, Kleckner N. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics. 1987;116(4):541–5.
Gietz RD, Schiestl RH. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc. 2007;2(1):31–4.
Zhou C, Yang H, Wang Z, Long GY, Jin DC. Comparative transcriptome analysis of Sogatella furcifera (Horvath) exposed to different insecticides. Sci Rep. 2018;8(1):8773.
Linak JA, Jacobson AL, Sit TL, Kennedy GG. Relationships of virus titers and transmission rates among sympatric and allopatric virus isolates and thrips vectors support local adaptation. Sci Rep. 2020;10(1):1–10.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25(4):402–8.
Satowa D, Fujiwara R, Uchio S, Nakano M, Otomo C, Hirata Y, Matsumoto T, Noda S, Tanaka T, Kondo A. Metabolic engineering of E. coli for improving mevalonate production to promote NADPH regeneration and enhance acetyl-CoA supply. Biotechnol Bioeng. 2020;117(7):2153–64.
Glicksberg BS, Amadori L, Akers NK, Sukhavasi K, Franzen O, Li L, Belbin GM, Ayers KL, Shameer K, Badgeley MA, et al. Integrative analysis of loss-of-function variants in clinical and genomic data reveals novel genes associated with cardiovascular traits. BMC Med Genomics. 2019;12(Suppl 6):108.
Merkling SH, Riahi H, Overheul GJ, Schenck A, van Rij RP. Peroxisome-associated Sgroppino links fat metabolism with survival after RNA virus infection in Drosophila. Sci Rep. 2019;9(1):2065.
Cao H, Xu Z, Wang J, Cigliano A, Pilo MG, Ribback S, Zhang S, Qiao Y, Che L, Pascale RM, et al. Functional role of SGK3 in PI3K/Pten driven liver tumor development. BMC Cancer. 2019;19(1):343.
Yang RM, Tao J, Zhan M, Yuan H, Wang HH, Chen SJ, Chen Z, de Thé H, Zhou J, Guo Y. TAMM41 is required for heart valve differentiation via regulation of PINK-PARK2 dependent mitophagy. Cell Death Differ. 2019;26(11):2430–46.
Strunov A, Boldyreva LV, Andreyeva EN, Pavlova GA, Popova JV, Razuvaeva AV, Anders AF, Renda F, Pindyurin AV, Gatti M, et al. Ultrastructural analysis of mitotic Drosophila S2 cells identifies distinctive microtubule and intracellular membrane behaviors. BMC Biol. 2018;16(1):68.
This work was supported by the Development of next-generation biorefinery platform technologies for leading bio-based chemicals industry project [NRF-2022M3J5A1056072], the Development of an integrated process to produce lignocellulosic biomass-derived fermentable sugars for next generation biorefinery project [NRF-2022M3J5A1056173], and the Bio & Medical Technology Development Program [NRF-2022M3A9B6082671] through the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science, ICT (MSIT). S.-H.S., S.Y.M., and J.Y.L. acknowledge funding from the Korea Research Institute of Chemical Technology through Core Program [SS2242-10].
The authors declare no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Squalene production of engineered strains in which the tHMG1 gene was integrated into a multicopy δ-sequence of the SQ00 genome. Figure S2. Differential interference contrast and confocal fluorescence microscopy images of the LDs in the LD metabolism-engineered strains. Table S1. List of plasmids and strains used in this study. Table S2. Sequence of N-degron tag. Table S3. Squalene production of the engineered strains in which Erg1 activity was partially inhibited by N-degron tag. Table S4. Squalene production of the LD metabolism-engineered strains.
About this article
Cite this article
Son, SH., Kim, JE., Moon, S.Y. et al. Metabolic recycling of storage lipids promotes squalene biosynthesis in yeast. Biotechnol Biofuels 15, 108 (2022). https://doi.org/10.1186/s13068-022-02208-9
- Lipid droplet
- Metabolic recycling
- Metabolic engineering
- Synthetic biology