Squalene is a natural product found in vegetable oils, fish oils, and human sebum. It is the main hydrocarbon component of human epidermal fats, accounting for about 10% by weight. Deep sea shark livers from the South Pacific, around Australia are the main source of squalene at present (Bhattacharjee et al., 2001). Squalene also exists in plant tissues, especially the oil in plant seeds, which is also an important industrial source of squalene (Gutierrez et al., 2014). Squalene is a lipophilic, acyclic triterpenoid compound formed from six isoprene units. It is the precursor of many biologically active compounds, including steroids and hopene (Katabami et al., 2015). Squalene itself also has useful applications, as a moisturizing ingredient in cosmetics (Kohno et al., 1995) and in pharmaceuticals (Reddy and Couvreur, 2009), based on its in anti-tumor (Rao et al., 1998), anti-fungal (Smith, 2000), and cholesterol-lowering (Kelly, 1999) effects. Currently, the main method of squalene production is extraction from plant materials, but the scale is limited by the long growing cycle of the plants (Lou-Bonafonte et al., 2018).

Compared with the conventional way of producing squalene from plant sources, microbial fermentation has the advantages in sustainability and greater environmental friendliness (Kim et al., 2019). For example, high titer squalene was obtained from the fungus, Yarrowia lipolytica by optimizing the fermentation medium (Tang et al., 2021), and 230 mg/L of squalene was obtained from Escherichia coli in flask culture by co-expressing mevalonate pathway with heterogeneous squalene synthase (Katabami et al., 2015). The cyanobacterium, Synechocystis, produced 5.1 mg/L of squalene, after introduction of a green algal squalene synthase (Pattanaik et al., 2020). However, few microorganisms possess a natural pathway for squalene synthesis. Enabling the production of squalene requires introduction of exogenous genes. Recently, the production of squalene with Saccharomyces cerevisiae has received considerable attention, because S. cerevisiae has a natural metabolic pathway for squalene synthesis, i.e., the mevalonate (MVA) pathway. In addition, S. cerevisiae is a well-developed eukaryotic producer microorganism and has been applied to produce many valuable plant natural products, including linalool, artemisinic acid and ginsenoside (Liu et al., 2013; Paramasivan and Mutturi, 2017a; Lian et al., 2018).

Many metabolic engineering strategies have been implemented to optimize squalene production by S. cerevisiae. The MVA pathway has been improved by overexpressing HMG-CoA reductase (HMGR) (Westfall et al., 2012). A truncated HMG-CoA reductase enzyme was overexpressed to eliminate feedback inhibition by ergosterol, (Polakowski et al., 1998). Metabolic flux optimization and NADH regeneration were achieved by co-overexpressing tHMG1 with POS5 (encoding NADH kinase), resulting in a 27.5-fold increase in squalene production (Paramasivan and Mutturi, 2017b). An engineered strain able to use ethanol as carbon source, with increased squalene synthesis, was obtained by overexpressing acetaldehyde dehydrogenase and alcohol dehydrogenase encoded by ADA and ADH2, respectively (Li et al., 2020b). Squalene production was also enhanced by increasing the storage capacity for hydrophobic compounds, by overexpressing DGA1, coding for diacylglycerol acyltransferase (Wei et al., 2018), and by cytoplasmic and peroxisomal engineering (Liu et al., 2020).

Practical industrial squalene production, however, still needs further enhancement of production by S. cerevisiae. Insufficient flux through the MVA pathway is an important limiting factor and the natural biosynthesis rate of the essential precursor, acetyl-CoA is insufficient for high squalene production. In this study, several strategies were implemented to enhance the production of squalene. To reduce the conversion of squalene into downstream compounds by squalene monooxygenase, 13 promoters were tested to replace the native promoter of the squalene monooxygenase gene, ERG1 (P_ERG1). To enhance the MVA pathway flux, IDI1 (isoprenoid diphosphate isomerase), and tHMG1 (HMG-CoA reductase, HMGR) were integrated at the multi-copy site Ty2 and NADH-HMG1 derived from Silicibacter pomeroyi was introduced. To supply more acetyl-CoA for squalene synthesis in the cytoplasm, TGL3, CIT2, FOX1, FOX2, FOX3, related to β-oxidation in peroxisomes, were overexpressed, and ACL from Y. lipolytica was heterologously expressed (Figure 1). In addition, a high-throughput screening method was established, to efficiently screen for high squalene-producing strains, resulting in a high-producing mutant, obtained by ARTP mutagenesis. The production of squalene was enhanced by 18.4%, permitting 8.2 g/L of squalene to be produced by a two-stage fermentation strategy, in a 5-L bioreactor.

S. cerevisiae is one of the most common microbial hosts for synthetic biology and metabolic engineering. It is a promising platform to produce squalene compared with natural resource extraction, or chemical synthesis (Polakowski et al., 1998; Thompson et al., 2014). In this study, the highest accumulation of squalene was achieved after the native promoter P _ERG1 was replaced by P _HXT1 . By randomly integrating tHMG1 and IDI1 into the multi-copy site Ty2, squalene production of 703.7 mg/L was obtained. Squalene production was further increased by 257 mg/L by increasing β-oxidation capacity. A high-throughput screening method was then established for screening high-efficiency squalene producers. After treatment by ARTP mutagenesis, a higher producing mutant strain was obtained, which accumulated 1470.9 mg/L squalene in a shake flask and 8.2 g/L in a 5 L fed-batch fermentation.

Weaker promoters of the squalene monooxygenase gene, ERG1, were tested to lower its expression level and minimize the conversion of squalene into downstream compounds. None of the weaker promoters resulted in significant squalene accumulation except P _HXT1 , which increased the squalene yield by 94.3% compared with the original strain. This indicated that the inducible promoter P _HXT1 was important for regulation of key genes related to yeast growth. P _HXT1 can also respond to the concentration of glucose (Ozcan and Johnston, 1995). During the growth stage of yeast, when the glucose concentration in the medium is relatively high, ERG1 is normally expressed, and the synthesis of sterols is sufficient to promote the rapid growth of the yeast cells. When the glucose in the culture medium is exhausted, expression of ERG1 is inhibited, thereby increasing the accumulation of squalene in the yeast cells (Zhao et al., 2017). Many reports have highlighted the importance of tHMG1 and IDI1 in increasing mevalonate (MVA) pathway flux and promoting squalene production (Liu et al., 2013). In this study, making various combinations of different copy numbers of tHMG1 and IDI1 showed that the production of squalene was proportional to their copy numbers. The integration of pathway genes in one or several copies, especially those encoding rate-limiting steps, is usual insufficient to maintain high metabolic fluxes (Maury et al., 2016). In addition, the expression of a multi-copy plasmid is unstable. The Ty2 element, with 34 insertions in the S. cerevisiae genome (Kim et al., 1998), was selected as the integration site for tHMG1 and IDI1.

Acetyl-CoA is a crucial precursor metabolite for terpene biosynthesis (Hellgren et al., 2020). In S. cerevisiae, acetyl-CoA metabolism is complex, because of its compartmentalized biosynthesis pathway and its involvement in various key cellular processes, including the tricarboxylic acid (TCA) cycle, biosynthesis of sterols, fatty acids, and polyketides (Nielsen, 2014). Strategies have been applied to enhance the supply of acetyl-CoA to the MVA pathway, such as introducing heterologous acetyl-CoA synthetase to increase acetyl-CoA (Shiba et al., 2007), removing bypass genes to eliminate competitive products (Lian et al., 2014), and establishing an efficient acetyl-CoA-producing bypass (Shiba et al., 2007; Li et al., 2020b). In addition, acetyl-CoA is a metabolite of fatty acid oxidation. In the peroxisome, it can be converted into citric acid and transported out of the peroxisome, through the glyoxylic acid cycle. The acetyl-CoA is converted to citric acid by citrate synthase (encoded by CIT2) and the resulting citric acid can be transported out of the peroxisomes to the cytoplasm (van Rossum et al., 2016). A heterologous citrate lyase, encoded by ACL1 and ACL2, was introduced, which converted citrate to acetyl-CoA, can promote the supply of acetyl-CoA to the cytoplasm. Genes involved in β-oxidation were also overexpressed. The citric acid generated by β-oxidation could be catalyzed to acetyl-CoA by the heterologous citrate lyase and further promoted squalene production. Furthermore, to enhance the growth of engineered strains, a diploid yeast was hybridized with two haploid, stress tolerant (e.g., to ethanol and starvation) yeasts, SQB4, and SQ6 (Tomova et al., 2019) (Supplementary Table S2).

The content of squalene in microorganisms is usually detected by high-performance liquid chromatography, which complicated and slow, because the cells must be disrupted, and extracted with organic solvent. It is, therefore, not suitable for high-throughput screening of high squalene-yield yeasts. Intracellular squalene is present as lipid droplets, which can be detected by Nile red staining (Greenspan et al., 1985; Chen et al., 2009; Sitepu et al., 2012; Rostron and Lawrence, 2017; Choi et al., 2018). After Nile red staining, the accumulation of squalene and lipid droplets in S. cerevisiae can be observed with a fluorescence microscope (Ta et al., 2012). This facilitated the establishment of a high-throughput screen, based on fluorescent Nile red staining of S. cerevisiae. The quantitative correlation between fluorescence intensity and squalene concentration from Nile red staining was analyzed with numerous cells of different squalene productivity. A high-throughput screening method was established, with a good linear relationship between squalene production and fluorescence intensity, by optimizing the concentration of Nile Red. Combined with the ARTP mutagenesis strategy and flow cytometry sorting technology (Figure 6), the screening efficiency was enhanced and a mutant strain was obtained, with squalene production increased by 18.4%.

Compared with previous reports, it appeared that the use of multi-copy site integration was an efficient way to increase MVA pathway flux and squalene production. However, the target sites for genomic integration have not been identified and there is a risk of DNA loss (Maury et al., 2016). A more accurate screening approach should be used after multi-copy site integration, such as whole genome sequencing. Furthermore, to promote the utilization of citric acid from β-oxidation, the glyoxylic acid cycle, which may be a competitive pathway, should be regulated to enhance squalene production. Except strengthening the supply of acetyl-CoA, overproduction of squalene can also be achieved by introducing a partial MVA pathway from acetyl-CoA to mevalonate in mitochondria (Zhu et al., 2021). It provides a new insight into the production of squalene and other terpenes in yeasts. Squalene distribution in yeast cells was visualized by Nile Red staining in this study, dense and large lipid droplets, spread over the whole cell were visualized by laser scanning confocal microscopy. Transporting squalene to the extracellular space appeared to be the main impediment to increased squalene accumulation. However, there are few reports on the transport of terpenoids in microorganisms, particularly hydrophobic terpenoids like squalene. The proteins most reported as being related to the transport of terpenoids are mainly ABC transporters, like SpTUR2 (Spirodela polyrrhiza) for the diterpene sclareol (Stukkens et al., 2005) and Snq2p for β-carotene (Sun et al., 2020). These findings indicated that the production of squalene could be further increased by discovering potential new transporters. Metabolic engineering has great potential to be applied to various cell factories to enhance the secretion of squalene.