High Production of Squalene Using a Newly Isolated Yeast-like Strain

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High Production of Squalene Using a Newly Isolated Yeast-like Strain Pseudozyma sp. SD301 Xiaojin Song,*,†,‡ Xiaolong Wang,∥ Yanzhen Tan,†,‡ Yingang Feng,†,‡ Wenli Li,∥ and Qiu Cui*,†,‡,§ †

Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, ‡Qingdao Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, and §Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao, Shandong 266101, People’s Republic of China ∥ Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao, Shandong 266003, People’s Republic of China S Supporting Information *

ABSTRACT: A yeast-like fungus, termed strain SD301, with the ability to produce a high concentration of squalene, was isolated from Shuidong Bay, China. The nucleotide sequence analysis of the internal transcribed spacer (ITS) region of SD301 indicated the strain belonged to Pseudozyma species. The highest biomass and squalene production of SD301 were obtained when glucose and yeast extracts were used as the carbon and nitrogen sources, respectively, with a C/N ratio of 3. The optimal pH and temperature were 6 and 25 °C, with 15 g L−1 of supplemented sea salt. The maximum squalene productivity reached 0.039 g L−1 h−1 in batch fermentation, while the maximum squalene yield of 2.445 g L−1 was obtained in fed-batch fermentation. According to our knowledge, this is the highest squalene yield produced thus far using fermentation technology, and the newly isolated strain Pseudozyma sp. SD301 is a promising candidate for commercial squalene production. KEYWORDS: marine fungi, optimization, Pseudozyma, squalene

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instability of production in certain plant species.9 Recently, major efforts have been made to identify alternative sources of squalene in microalgae and microorganisms. The yield of squalene is approximately 0.43−340.5 mg L−1 dry cell weight (DCW) from yeasts15−17 and a lower productivity than 0.72 mg g−1 DCW from thraustochytrids.18,19 Currently, several Aurantiochytrium strains, such as strain 18W-13a20−22 and strain PQ6,23 can produce squalene at a yield of approximately 1 g L−1. However, the squalene productivity is still very low for commercial use. In this study, we screened various environmental samples for squalene-producing microorganisms with high yield and isolated several squalene producers. Among them, we identified a novel yeast-like strain that possessed the highest squalene productivity thus far. This isolate, termed Pseudozyma sp. SD301, proved its strong potential in commercial squalene production.

INTRODUCTION Squalene (2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22hexane, C30H50) is a linear polyunsaturated aliphatic hydrocarbon, which is an intermediate for the biosynthesis of bile acids, ergosterol, and steroids.1 In addition, squalene has a natural antioxidant effect in a model of lipid peroxidation of liposomes that protects cells from free radicals and reactive oxygen species (ROS).2,3 Recent epidemiological studies have indicated that squalene has effective antitumor activities, which can strengthen the immunity of humans and decrease the risk of various cancers, such as colon, lung, and skin tumorigenesis.4,5 In experiments with rats, a significant reduction in the level of lipid peroxidation in the heart tissue is observed by a given combined administration of squalene and polyunsaturated fatty acids.6 Moreover, squalene also has a cardioprotective effect on experimentally induced myocardial infarction.7,8 Currently, squalene is used as a moisturizing agent and an emollient in the cosmetic industry, and it has garnered more and more attention for its potential applications in pharmaceuticals and medical sectors.3 The extensive use of squalene in industry is hampered by limited resources and its relatively high price. Currently, the commercial sources of squalene are the liver oil of deep sea sharks and vegetable oils.9 Squalene is the main ingredient in the liver oil of sharks, which could reach up to a percentage of 60%.10 Some plant seed oils also have high squalene levels, such as peanut oil (1.28 g kg−1),2 amaranth oil (2.15 g kg−1),11 and olive oil (5.99 g kg−1).12 However, the continuous supply and future availability of squalene are uncertain because of the decreasing number of sharks, increasing environmental pollutants as well as an unpleasant fishy smell,13,14 and the © XXXX American Chemical Society

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MATERIALS AND METHODS

Isolation and Screening of High-Squalene-Producing Strains. Pseudozyma strains were obtained from seawater, soil, and leaf samples of the mangrove ecosystem in Shuidong Bay, Guangdong, China. The samples were incubated on KMV+-medium-containing agar plates (glucose, 1.0 g L−1; gelatin hydrolysates, 1.0 g L−1; yeast extract, 0.1 g L−1; peptone, 0.1 g L−1; and agar, 12 g L−1; in 1 L of natural seawater containing penicillin G, 50 ppm; and streptomycin Received: July 11, 2015 Revised: September 6, 2015 Accepted: September 9, 2015

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DOI: 10.1021/acs.jafc.5b03539 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry sulfate, 50 ppm) at 25 °C in the dark. After 90 h of incubation, the monocolonies on the surface of the plates were transferred onto KMV+ medium several times to purify the strains. These isolated strains were kept on a KMV+ medium (without antibiotics) as monoclonal cultures, and then we determined their squalene content using gas chromatography. Molecular and Phylogenetic Analyses. The genomic DNA of selected strains was extracted using a Universal Genomic DNA extraction kit (Takara DV811A, Dalian, China), according to the protocol of the manufacturer. The internal transcribed spacer (ITS) region was amplified using the ITS4 primer P1 (5′TCCTCCGCTTATTGATATGC-3′) and the ITS5 primer P2 (5′GGAAGTAAAAGTCGTAACAAGG-3′). The polymerase chain reaction (PCR) products were visualized by agarose gel electrophoresis and purified using the TIANgel Midi Purification Kit (TIANGEN DP209, Beijing, China). The purified PCR products were cloned into the pGEM-T Easy vector (Promega) and then were sequenced by Takara Co., Ltd. The resulting sequence data were searched using NCBI BLAST, and the sequences were aligned with the ClustalX program. A phylogenetic tree was inferred using the Minimum Evolution method of MEGA, version 5.0. The tree reliability was evaluated by bootstrap analysis of 1000 replicates. Strains and Cultivation. Strain SD301 was isolated by the methods mentioned above and cultivated in 50 mL of GPY medium (6% glucose, 1% peptone, and 1% yeast extract) containing 1.5% artificial sea salt in a 250 mL flask shaken at 200 rpm and 25 °C. Optimization of Culture Conditions. To investigate the effects of various cultivation media, eight carbon sources (6% of glucose, Dfructose, D-xylose, maltose, sucrose, lactose, glycerol, and starch) and seven nitrogen sources (2% of yeast extract, peptone, tryptone, urea, ammonium sulfate, sodium glutamate, and sodium nitrate) were tested. Tryptone (2%) was used as the nitrogen source for the carbon source testing, while glucose (6%) was consistently used as the carbon source when the nitrogen source testing was carried out. To investigate the C/N ratio effect on the cell growth and squalene yield of strain SD301, the constant concentration of the C source of 6% and various N source concentrations were used. The effects of varying concentrations (0−60 g L−1) of sea salt were also studied using the optimized medium. To examine the effects of various growth parameters, various pH values (pH 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0) and different temperatures (15, 20, 25, 30, and 35 °C) were used to cultivate Pseudozyma sp. SD301. All tests were carried out in 250 mL flasks with 50 mL medium shaken at 200 rpm and 25 °C for 5 days. Batch and Fed-Batch Fermentation Experiments for Squalene Production. A batch fermentation experiment was performed in a 5 L Biostat B plus bioreactor, which was equipped with controllers for pH, temperature, agitation, and dissolved oxygen (DO) concentration. Batch cultures were carried out in 3.5 L of production medium. The temperature was maintained at 25 °C. The agitation speed automatically varied from 300 to 800 rpm at a fixed air flow rate of 1.2 vessel volumes per minute (vvm) to maintain the DO at 20% air saturation. The pH was maintained at 6.0 by adding 2 mol L−1 NaOH or 2 mol L−1 HCl. To control foam formation, 1 mL of antifoam was added at the beginning of the run. Samples for off-line determination of the glucose concentration, biomass, and squalene yield were withdrawn every 6 h until the end of the fermentation (48 h). Another fed-batch fermentation experiment was also performed in the 5 L Biostat B plus bioreactor, with the same conditions of the batch fermentation, except that intermittent glucose feeding was supplied to maintain the residual glucose concentration at approximately 5 to 25 g L−1 by feeding a 40% (w/v) glucose stock solution. The samples were withdrawn every 10 h until the end of the fermentation (100 h). The initial fermentation medium contained 60 g L−1 glucose and 20 g L−1 yeast extract in artificial seawater (artificial sea salt concentration of 15 g L−1). This medium also contained a vitamin solution (2 mL L−1). The vitamin solution was filter-sterilized (0.22 μm) and

contained 100 mg/L thiamine, 10 mg/L biotin, and 50 mg/L cyanocobalamin. Biomass Determination and Glucose Assay. The biomass of Pseudozyma sp. SD301 was expressed as DCW. Samples (5 mL) were collected and centrifuged at 7000g at 4 °C for 10 min, and they were then freeze-dried to a constant weight at −50 °C for approximately 60 h. The residual glucose in the fermentation medium was analyzed by a biosensor equipped with a glucose oxidase electrode (SBA-40E, Institute of Biology, Shandong Academy of Sciences, Jinan, China). Observation Using Confocal Laser Scanning Microscopy (CLSM). To investigate the intracellular localization of squalene in Pseudozyma sp. SD301, the observation of in vivo fluorescence imaging of the cells were carried out using CLSM (FluoView FV1000, Olympus, Tokyo, Japan). First, the cells were dyed by nile red using the method mentioned by Morita et al.24 In details, 500 μg mL−1 nile red was prepared in acetone and stored at 4 °C as the stock solution. Then, the stock solution was added to the culture with a final concentration of 0.1 μg mL−1 and incubated at 37 °C for 15 min. Second, the stained cells were observed by CLSM, and the excitation and emission wavelengths were 559 and 612 nm, respectively. The fluorescent images were obtained at 1000× magnification. Lipid Extraction and Fatty Acid and Squalene Content Analyses. The total lipid content was estimated using a modified miniaturized Bligh−Dyer method.25 The harvested cells were extracted into 30 mL of chloroform/methanol (2:1, v/v) at room temperature. The lipid extract was dried over anhydrous Na2SO4, and the solvent was removed by evaporation. The total lipid was converted into fatty acid methyl esters (FAMEs), and the fatty acid composition was analyzed using an Agilent 78905975 gas chromatography−mass spectrometry (GC−MS) system (Agilent Technologies, Inc., Santa Clara, CA).26 Squalene was isolated from the total lipid by saponification,27 with some modifications. Samples (5 mL, V1) were freeze-dried and used to extract the total lipid by the methods mentioned above. The extracted total lipid was transferred into 10 mL of 10% KOH−ethanol solution and incubated at 60 °C for 1 h with refluxing for saponification. Then, 4 mL of distilled water was added, and the unsaponifiable compositions were extracted with 2 mL (V2) of n-hexane. Afterward, squalene was separated by Agilent 7890A GC with a 7650A automatic liquid sampler using a HP-5, 30 m × 320 μm × 0.25 μm column; meanwhile, nitrogen was used as a carrier gas. The initial oven temperature was 100 °C and was maintained for 1 min, and then the temperature was raised to 300 °C for 15 °C min−1 and, finally, maintained at 300 °C for another minute. Peak detection was performed by a flame ionization detector. The temperature of the injection port and flame ionization port was 300 °C. The injection volume was 1 μL, and the split ratio was 1:10. The squalene concentration (X, μg mL−1) was further quantified using an external standard method. The standard curve (Figure S1) was made from a set of concentrations of commercial squalene standard (>99% squalene, J&K Chemical) by GC analysis. The squalene production (P, g L−1) was calculated by eq 1.

P=X

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V2 × 10−3 V1

(1)

RESULTS AND DISCUSSION Isolation and Identification of Pseudozyma sp. SD301. Samples obtained from a mangrove ecosystem were screened for squalene-producing microorganisms. Almost 100 purified strains were isolated as colonies on the KMV+ medium plates and microscopically observed. The biomass (DCW) of the strains ranged from 5.29 to 27.61 g L−1 when cultivated in the GPY medium, and the squalene contents (percent of DCW) and squalene yields ranged from 0.4 to 5.6% DCW and from 0.1 to 0.9 g L−1, respectively. Among these tested strains, SD301 showed the highest levels of squalene content and yield. On the basis of morphological B

DOI: 10.1021/acs.jafc.5b03539 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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features of the genus Pseudozyma.28 Subsequently, the ITS region of this strain was determined, and SD301 was confirmed to be a Pseudozyma strain by phylogenetic analysis using the minimum-evolution (ME) tree (Figure 2). Hence, the newly isolated strain was termed Pseudozyma sp. SD301. To find the optimal conditions for squalene production, we further investigated the cell growth and squalene production on various carbon and nitrogen sources and under various temperatures, pH, and osmotic stresses, all of which are necessary for the optimization of the culture conditions. Cell Growth and Squalene Production on Various Carbon and Nitrogen Sources. As shown in Figure 3a, glucose, fructose, xylose, maltose, sucrose, starch, glycerol, and lactose were used as carbon sources to investigate the cell growth and squalene yield of Pseudozyma sp. SD301. The monosaccharide hexoses, such as glucose and fructose, could be well used by Pseudozyma sp. SD301 with a biomass of 24.06 and 19.09 g L−1, respectively, but pentoses (such as xylose) and polysaccharides (e.g., starch) were not efficient substrates for cell growth. Sucrose was a good carbon source for Pseudozyma sp. SD301 growth, while maltose, another disaccharide, was not optimal. At the same time, glycerol and lactose were also very suitable for its growth. In contrast, only three carbon sources, glucose, sucrose, and lactose, were fit for optimal squalene production by Pseudozyma sp. SD301, with the best squalene yield of 0.79 g L−1 (Figure 3a). These results were in agreement with previous data obtained for Pseudozyma sp. JCC207.15 Peptone, yeast extract, tryptone, urea, sodium glutamate, ammonium sulfate, and sodium nitrate were tested as organic or inorganic nitrogen sources for the growth of Pseudozyma sp. SD301 (Figure 3b). When yeast extract was used as a nitrogen source, the highest biomass and squalene production (28.32 and 0.788 g L−1, respectively) were obtained, followed by tryptone, peptone, sodium glutamate, ammonium sulfate, urea, and sodium nitrate. According to our results, organic nitrogen sources, such as yeast extract, tryptone, and peptone, were more suitable for the cell growth and squalene production than inorganic nitrogen sources because these organic nitrogen sources had, in contrast to inorganic nitrogen sources,

observations, SD301 formed a yeast-like vegetative cell that was ovoid to cylindrical and produced fusiform blastoconidia (Figure 1). These morphological characteristics were common

Figure 1. Morphology of Pseudozyma sp. SD301 (a, light microscope, 400× magnification; b, scanning electron microscope, 3500× magnification).

Figure 2. Phylogenetic relationship of Pseudozyma species based on the aligned ITS sequences. The tree was constructed using the ME method and 1000 bootstrapped replicates in MEGA5.0. C

DOI: 10.1021/acs.jafc.5b03539 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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that the high C/N ratio might lead to relative nitrogen hunger (or other nutrient hunger, e.g., phosphorus and sulfur), which limits cell growth and increases the biosynthesis of other lipids (such as fatty acids and triglyceride). Effects of the Sea Salt Concentration and Culture Temperature on Cell Growth. The effects of the sea salt concentration on cell growth and squalene production were examined in a range of 0−6%. Pseudozyma sp. SD301 showed a wide salinity tolerance. As shown in Figure 4a, the optimum salt

Figure 3. Effects of (a) carbon sources, (b) nitrogen sources, and (c) C/N ratio on the cell growth and squalene yield in Pseudozyma sp. SD301.

additional growth factors, such as vitamins and coenzymes, which might be necessary for squalene biosynthesis. These results were slightly different from the results of Pseudozyma sp. JCC207,15 which could also produce squalene with inorganic nitrogen sources. It is well-known that the C/N ratio of the culture medium dramatically affects the growth of microorganisms. A high C/N ratio is effective for increasing the lipid content in cells.29 Therefore, the effects of the C/N ratio on the cell growth and squalene yield of strain SD301 were examined (Figure 3c). The cell growth increased to the maximum level by increasing the C/N ratio to 3. The highest squalene yield of 0.756 g L−1 was also obtained at this C/N ratio. A further increase of the C/N radio showed a negative effect on both the cell growth and squalene yield. When the C/N ratio reached 7.5, the biomass and squalene production decreased to 19.53 and 0.632 g L−1, respectively. This phenomenon could be explained by the fact

Figure 4. Effect of (a) artificial sea salt concentration, (b) temperature, and (c) initial pH on cell growth and squalene yield in Pseudozyma sp. SD301.

concentration was 15 g L−1, with a biomass and squalene yield of 26.25 and 0.847 g L−1, respectively. A slight change in the cell growth was observed in the range of 0.75−3%. Many microorganisms living in the mangrove ecosystem show euryhalinity30,31 because the salinity of this area often sharply changes as a result of the afflux of fresh water and evaporation. The effects of the temperature on biomass and squalene yield were tested from 15 to 35 °C. As shown in Figure 4b, there were only slight fluctuations in biomass and squalene yield between the temperatures of 25 and 30 °C. The biomass production was the highest (26.23 g L−1) at 25 °C. However, when the temperature was raised to 35 °C, the biomass D

DOI: 10.1021/acs.jafc.5b03539 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 5. Observation of Pseudozyma sp. SD301 using CLSM for locating the lipid droplets and squalene accumulation during cultivation.

decreased to 21.37 g L−1 and the squalene yield also decreased significantly, from 0.811 g L−1 at 25 °C to 0.555 g L−1 at 35 °C. The variation in squalene yields may be the result of the biosynthesis of ergosterol and steroids, which are vital structural and regulatory components in eukaryotic cells. Effects of Initial pH on Cell Growth. Because the pH value of the medium profoundly affects cell membrane functions, cell metabolism, and the uptake of nutrients and product biosynthesis, the initial pH values of the medium tested in this study ranged from 4.0 to 9.0. The results showed that strain SD301 had a wide adaptability of initial pH (4.0−9.0) for growth and squalene accumulation (Figure 4c). The variation of biomass and squalene yields in the tested pH ranges ranged from 21.70 g L−1 (pH 9.0) to 26.90 g L−1 (pH 6.0) and from 0.625 g L−1 (pH 4.0) to 0.820 g L−1 (pH 6.0), respectively. The optimal pH for biomass and squalene yield was 6.0. The wide pH adaptability of Pseudozyma sp. SD301 may due to its adaptation to the mangrove ecosystem, in which the pH is constantly changing with tidal actions. Microscopic Observation and the Fatty Acid Composition of Pseudozyma sp. SD301. From the fluorescence imaging of the cells (Figure 5), the lipid droplets containing squalene were distinctly observed at the both ends of the cells and were accumulated along with the cell growth. This phenomenon was coincident with that in Saccharomyces cerevisiae.16,17 The fatty acid composition of SD301 was shown in Table 1, and the main ingredients were the fatty

acids with 16 or 18 carbons, such as C16:0, C18:0, C18:1, and C18:2. Along with the cell growth, the content of saturated fatty acids in cells decreased and the content of unsaturated fatty acids reached up to over 51%. Batch and Fed-Batch Fermentation Experiments for Squalene Production. Using the optimized culture conditions described above, the biomass, squalene production, and glucose consumption were examined during batch and fedbatch cultivation. Glucose was exhausted after 42 h of batch fermentation, reaching the highest biomass and squalene production of 30.24 and 1.649 g L−1, respectively (Figure 6a). The conversion rate from glucose to biomass was approximately 50.4%, and the squalene productivity reached 0.039 g L−1 h−1. In contrast, for the fed-batch fermentation, a total glucose concentration of 143 g L−1 was consumed and the maximum DCW was 71.53 g L−1 with a squalene yield of 2.445 g L−1 at 80 h (Figure 6b). In comparison to the batch fermentation, both the glucose conversion rate and the squalene productivity were slightly decreased. This result might be caused by the increasing C/N ratio during glucose feeding, which could influence cell growth and increase fatty acid and triglyceride biosynthesis. Some strains of Aurantiochytrium (Schizochytrium or Thraustochytrium) were used to produce squalene with the final yield of approximately 1 g during 96 h of fermentation. In comparison to these strains, Pseudozyma sp. SD301 had superior squalene productivity and higher squalene yield (Table 2). The squalene yield of 2.445 g L−1 by Pseudozyma sp. SD301 was 2 times greater than the reported highest yield of Aurantiochytrium sp. 18W-13a (1.29 g L−1), and the squalene productivity was approximately 3 times higher than that of Aurantiochytrium strains. In summary, this study characterized a newly isolated yeastlike strain, Pseudozyma sp. SD301, which is a promising candidate for commercial squalene production. The results illustrated that the optimized medium for squalene production should contain 60 g L−1 glucose, 20 g L−1 yeast extract, and 15 g L−1 artificial sea salt concentration, with the appropriate cultivation conditions of pH 6.0 and 25 °C.

Table 1. Fatty Acid Profiles of Pseudozyma sp. SD301 content (% TFA) fatty acid

24 h

72 h

120 h

C14:0 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2 C20:0

2.03 30.06 2.32 2.44 24.12 23.21 13.87 1.83

1.57 26.46 2.09 2.77 20.99 29.22 14.58 2.28

1.34 24.89 1.45 1.51 15.97 35.94 16.74 2.14 E

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Figure 6. Fermentation experiments of Pseudozyma sp. SD301 in a 5 L bioreactor: (a) batch fermentation experiment and (b) fed-batch fermentation experiment: (open squares) residual glucose, (closed diamonds) biomass, and (closed triangles) squalene yield.

Funding

Table 2. Comparison of the Squalene Yield by the Strains Mentioned in This Study strain Aurantiochytrium sp. 18W-13a (2011) Aurantiochytrium sp. 18W-13a (2012) Thraustochytrium (2013) Schizochytrium mangrovei PQ6 (2014) Pseudozyma sp. JCC207 (2008) Pseudozyma sp. SD301

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squalene yield (g L−1) 1.29

squalene productivity (g L−1 h−1) 0.014

0.9 ≈1

This work was financially supported by the National Natural Science Foundation of China (41306132) and the National High Technology Research and Development Program of China (863 Program, 2014AA021701).

reference 20

Notes

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The authors declare no competing financial interest.

0.012

22

0.992−1.019

0.011

23

0.341

0.004

15

2.445

0.039

this study

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b03539. Standard curve of squalene concentrations (Figure S1) (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

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DOI: 10.1021/acs.jafc.5b03539 J. Agric. Food Chem. XXXX, XXX, XXX−XXX