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Cell surface changes that advance the application of using yeast as a food emulsifier
Food Chemistry 315 (2020) 126264
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Cell surface changes that advance the application of using yeast as a food emulsifier
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Shinsuke Nerome, Masaya Onishi, Daiki Saito, Ayano Mizobuchi, Tatsuya Ando, Yui Daira, ⁎ Azusa Matsumoto, Yoshihiro Ojima, Masayuki Azuma Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, Sugimoto 3-3-138, Sumiyoshi-ku, Osaka 558-8585, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Emulsification Yeast Cell wall Mannan Emulsifier
A previous study revealed that Saccharomyces cerevisiae mcd4Δ, a cell wall mutant with a defect in the synthesis of the glycosylphosphatidylinositol anchor, has a strong macrophage activation ability. In this study, remarkable emulsion formation after cell suspensions of mcd4Δ and anp1Δ (which exhibit an extreme reduction of mannan) were mixed with oil was found. Moreover, the relationship between cell wall mutation and emulsion formation was investigated, suggesting that och1Δ with a defect in the formation of N-linked glycans also had a strong emulsification ability and that high molecular weight materials released from the cells were involved in emulsion formation. Furthermore, two strains (asc1Δ and scp160Δ) with a strong emulsification ability without a large decrease in mannan content were also found from the wide screening of strains that exhibit an emulsifying activity using more than 5000 gene-deficient strains. These results provide valuable information for the development of a yeast-derived emulsifier.
1. Introduction Emulsification is a procedure frequently performed in the food and cosmetics industries in which emulsifiers are used. In general, chemically synthesized emulsifiers are often used; however, some naturally derived emulsifiers like casein are also used, but not in large quantities. Yeast is presumably a natural emulsifier. If yeast can have both fermentative and adequate emulsification abilities, there will be no need to add chemically synthesized emulsifiers to many food products that use yeast, such as bread. There are a few reports on the ability of yeast to form emulsions; for example, mannoproteins obtained from β-1,3glucanase or physical treatment of cells have been reported to have an emulsifying effect (Cameron, Cooper, & Neufeld, 1988; De Iseppi et al., 2019; Dikit, Maneerat, Musikasang, & H-kittikun, 2010; Li & Karboune, 2019). In addition, research has been conducted on the oil-in-water emulsion formed by Saccharomyces cerevisiae cells for use in oil production (Meirelles, da Cunha, & Gombert, 2018); however, so far, there is no research on how to enhance the emulsification ability of yeast. In order to use yeast as an emulsifier in food, it is necessary to overcome the cost barrier of preparation; developing yeast suitable for this use is one of the solutions.
The cell wall of S. cerevisiae comprises mannoproteins, β-1,3-glucan, β-1,6-glucan, and chitin, which account for approximately 30–50%, 30–45%, 5–10%, and 1.5–6%, respectively, of the dry cell wall weight (Klis, Boorsma, & De Groot, 2006). The majority of mannoproteins are covalently coupled to cell wall polysaccharides and are divided into two types: glycosylphosphatidylinositol cell wall proteins (GPI-CWPs) and alkali-sensitive linkage CWPs. GPI-CWPs are attached to β-glucans through GPI anchors. These proteins contain high-mannose N-linked glycans and are localized in the outermost layer of the cell wall, covering the cells with mannan (Klis et al., 2006; Lesage & Bussey, 2006; Orlean, 2012; Cabib & Arroyo, 2013). In addition, there are noncovalently bound CWPs, such as heat shock proteins and glycolytic enzymes (López-Ribot & Chaffin, 1996; Delgado et al., 2001), which are released from the cells by a reducing agent under weak alkaline conditions. These noncovalently bound proteins are trapped inside the cell wall or weakly ionically bound to other CWPs. The physiological functions of β-glucans are well known (Novak & Vetvicka, 2008), and the immunomodulatory effects of S. cerevisiae βglucans have been reported previously (Young, Ye, Frazer, Shi, & Castranova, 2001; Ikeda et al., 2008; De Smet, Allais, & Cuvelier, 2014). The immunomodulatory effects of whole S. cerevisiae cells have been
Abbreviations: GPI, glycosylphosphatidylinositol; CWP, cell wall protein; TNF-α, tumor necrosis factor-α; MATa, mating type a; MATα, mating type α; WT, wild type; PBS, phosphate-buffered saline; YPD, yeast extract peptone dextrose; PBSS, phosphate-buffered saline containing sorbitol; MWCO, molecular weight cutoff ⁎ Corresponding author. E-mail address: [email protected] (M. Azuma). https://doi.org/10.1016/j.foodchem.2020.126264 Received 3 October 2019; Received in revised form 10 January 2020; Accepted 17 January 2020 Available online 21 January 2020 0308-8146/ © 2020 Elsevier Ltd. All rights reserved.
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2.4. Analyses of substances released from yeast cells
the focus because of their beneficial effects on host immunity through fermented foods containing yeast (Sakai et al., 2007; Takada, Nishino et al., 2014, Takada, Mizobuchi et al., 2014). Previous studies have reported a large decrease in cell wall mannan in the mutant strain mcd4Δ, which is defective in the synthesis of GPI anchors necessary for fixing mannoproteins to β-glucans (Maneesri et al., 2005). These mutant mcd4Δ cells strongly activated mouse macrophages through direct contact in vitro and induced high secretion of tumor necrosis factor-α (TNF-α). Mouse immune cells were also activated in vivo by contact with mcd4Δ cells (Sakai et al., 2007). In this study, the analysis of mcd4Δ processes showed remarkable emulsion formation when cell suspensions were mixed with oil. With this result as a trigger, focus was placed on gene mutations that enhance the emulsification ability. The effects of various genetic mutations were examined, and cell surface changes that lead to the high emulsifying activity of cells were found. In addition, it was confirmed that CWPs easily released from yeast cells are involved in emulsion formation.
The OD600 value of mcd4Δ cells after cultivation for 48 h was adjusted to 15 using PBSS, and a 20 mL cell suspension was centrifuged at 3000×g for 5 min, after which the pellet was resuspended in 20 mL of PBS. The suspension was then vortexed and centrifuged at 3000×g for 5 min. The supernatant (10 mL) was filtered through an Amicon® Ultra Centrifugal Filter (10,000 or 100,000 molecular weight cutoff, MWCO), and any residual material that did not pass through the filter was diluted to its original volume using PBS. The emulsifying activities of the unfiltered material (top chamber) and the filtrate (bottom chamber) were measured as described above. 2.5. Emulsion formation by substances released from yeast cells after treatment with β-1,3-glucanase Wild type (WT) cells were cultured in a YPD medium at 30 ℃ overnight, harvested by centrifugation, and washed with 66 mM PBS (pH 7.5) containing 1.2 M sorbitol. The OD600 value was adjusted to either 1.0 or 3.0 with the buffer. β-1,3-Glucanase (Zymolyase-100T) was added to the cell suspension (4 mL) to 10 units/mL of the final concentration. The suspension was then incubated at 35℃ for 2 h and centrifuged at 700×g for 5 min. The pellet was washed with the buffer and then resuspended in 4 mL of buffer. The supernatant or pellet suspension (2 mL) was mixed with 1 mL of kerosene, and emulsion formation was assessed.
2. Materials and methods 2.1. Strains and media S. cerevisiae BY4741 (mating type a [MATa] his3Δ1 leu2 Δ 0 met15Δ0 ura3 Δ 0) was used as the parental strain (Brachmann et al., 1998), and each knockout strain (BY4741, orf Δ::kanMX4) (Winzeler et al., 1999) was purchased from GE Healthcare UK Ltd. (Buckinghamshire, UK) and used for screening. In this paper, for example, mcd4 gene-deficient strain is denoted as mcd4Δ. For mcd4Δ (MATa) only, the strain that was obtained previously from the heterozygous strain was used (Maneesri et al., 2005). S. cerevisiae BY4742 (mating type α [MATα] his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) and each knockout strain (BY4742, orf Δ::kanMX4) was used to confirm the effects of gene deletions selected in the screening. A yeast extract peptone dextrose (YPD) medium (1% [w/v] yeast extract, 2% [w/v] peptone, and 2% [w/v] glucose) was used to culture the yeast strains. The yeast extract was purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). Sorbitol (0.6 M) was added to the YPD medium when osmotic support was necessary; this medium was denoted as YPDS.
2.6. Screening of yeast strains with emulsifying activity First, deletion strains (MATa) of the Yeast Knockout deletion collection were screened. In order to make the operation as easy as possible, the cells were cultured on YPDS agar at 30 ℃ for 48 h, and approximately equal amounts were visibly judged and gathered using a toothpick from single colonies on the plates and suspended in PBS. A 0.5 mL suspension was mixed with 0.5 mL of kerosene in a 1.5 mL Eppendorf tube and incubated at 30 ℃ for 1 h to observe emulsion formation. Emulsification similar to that of mcd4Δ was evaluated as + +, and the emulsification ability of the tested strains was divided into four categories ranging from −to ++. Strains that exhibited ++ emulsification ability were selected, and the effects of those deletion genes were confirmed from the emulsification ability of gene deletion strains of MATα. In order to further investigate the details of emulsion formation with the eight selected strains, the cells were cultured at 30 ℃ for 48 h and harvested by centrifugation at 3000×g for 5 min, and the OD600 value was adjusted to 0.5 with PBSS. A YPDS liquid medium was used for the culture. The cell suspension (50 mL) was centrifuged, and the pellet was resuspended in 7 mL of PBS. The cells were then centrifuged again, and the supernatant was collected and lyophilized. The lyophilized material was resuspended in 1.1 mL of pure water; a 1 mL sample was mixed with 1 mL of kerosene, and emulsion formation was observed. Emulsification similar to that of mcdΔ was assessed as +++, and the emulsification ability of mutants was divided into five categories ranging from − to +++. Mutants that showed an emulsification ability of +++ in both MATa and MATα were selected.
2.2. Emulsion formation by yeast cells Each strain was cultured in a YPDS liquid medium at 30 ℃ for 48 h, and the cells were harvested by centrifugation at 3000×g for 5 min. In order to measure emulsion formation by yeast cells, the optical density at 600 nm (OD600) was adjusted to 0.7 using phosphate-buffered saline (PBS) containing 0.6 M sorbitol (PBSS), and a 5 mL cell suspension was centrifuged at 3000×g for 5 min. The pellet was then resuspended in 5 mL of PBS, and 1 mL of either kerosene or tetradecane was added. After vortexing for 30 s, the suspension was incubated at 30 ℃ for 1, 24, or 48 h and photographed after each time period. The image of micelles immediately after mixing tetradecane with the mcd4Δ cell suspension cultured for 48 h was observed under an Olympus BH50 microscope (Olympus, Tokyo, Japan). 2.3. Emulsion formation by substances released from yeast cells
2.7. Macrophage activation by contact with yeast cells Each strain was cultured in a YPDS liquid medium at 30 ℃ for 48 h. After harvesting, the OD600 value was adjusted to 0.7 using PBSS. The cell suspension (5 mL) was then centrifuged at 3000×g for 5 min, and the pellet was resuspended in 5 mL of PBS and centrifuged again at 3000×g for 5 min. The supernatant (5 mL) and 1 mL of kerosene were mixed, and emulsion formation was assessed as described above. After the last centrifugation, the precipitate was resuspended in 5 mL of PBS and mixed with 1 mL of kerosene, after which emulsion formation was assessed.
Macrophage activation by contact with yeast cells was measured according to a previously described method with some modifications (Sakai et al., 2007). Yeast cells cultured at 30 ℃ for 24 h in a YPDS medium were harvested and washed twice with PBSS. mcd4Δ cells were cultured for 48 h because of their slow growth. The cells were then fixed in 70% (v/v) ethanol and lyophilized. The dried cells were resuspended in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (ICN 2
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Biomedicals, Aurora, OH, USA). The mouse macrophage line RAW264.7 (ATCC TIB-71) was also precultured in RPMI-1640, washed twice with medium, counted, and resuspended in fresh medium at 0.5 × 106 cells/mL. The cells were then cultured for 24 h, the culture medium was removed, and 0.5 mL of new medium containing yeast cells at a final concentration of 100 μg/mL was added. After adding the yeast samples to the macrophages, the solution was incubated in 5% CO2 at 37 ℃ for 6 h and centrifuged, and the supernatant was recovered. The levels of mouse TNF-α in the supernatants were measured using enzyme-linked immunosorbent assay according to the manufacturer’s instructions (R&D Systems, Inc., Minneapolis, MN, USA). In addition, baker’s yeast-derived insoluble β-glucan (Oriental Yeast Co., Ltd.) was used as the control. The final concentration was 50 μg/mL. 2.8. Ratio of mannose to glucose in the cell wall The amounts of glucose and mannose in the cell wall were evaluated according to a previously reported method (Dallies, Francqois, & Paquet, 1998; Takada, Nishino et al., 2014). The cells were cultured at 30 ℃ for 24 h, harvested by centrifugation at 3000×g for 5 min, and washed twice with PBSS. mcd4Δ cells were cultured for 48 h in a YPDS liquid medium. The cells were then broken using glass beads on ice, and cell walls were collected by centrifugation at 3800×g for 5 min and then freeze-dried. Then, 3 mg of the collected cell walls was suspended in 75 μL of 72% (w/w) H2SO4 and kept at room temperature for 3 h. The obtained slurry was diluted to 1 mL with pure water and heated at 100 ℃ for 4 h. The suspension was then cooled on ice and neutralized with saturated Ba(OH)2. The supernatant was collected by centrifugation at 3800×g for 5 min and kept at 4 ℃ overnight. The precipitate was removed by centrifugation, and the supernatant was freeze-dried. The amounts of glucose and mannose were measured using a highperformance liquid chromatograph equipped with an RID-6A detector (Shimadzu Corp., Kyoto, Japan) and a Rezex RPM-Monosaccharide column (300 × 7 mm; Phenomenex, Torrance, CA, USA). Milli-Q water (Millipore Corp., Bedford, MA, USA) was used for the elution. The flow rate and column temperature were 1.0 mL/min and 78 ℃, respectively.
Fig. 1. Emulsion formation by mixing oil with an mcd4Δ cell suspension cultured for 24 h (A) and a microscopic image of micelles formed by mixing kerosene and an mcd4Δ cell suspension (B).
2.9. Statistical analysis In order to measure the ratio of mannose to glucose in the cell wall as well as macrophage activation by contact with yeast cells, all results are shown as the mean ± standard deviation of independent experiments (n ≥ 3). Welch’s t-test was used to compare two groups. Differences were assessed using a two-sided test with an ⍺-level of 0.05. Significant differences from WT were indicated by an asterisk (*P < 0.05).
3.2. Emulsion formation by anp1Δ cells Mannan staining using Con A-FITC showed that the amount of mannan in the mcd4Δ cell wall was extremely low (Maneesri et al., 2005); however, no quantitative evaluation was performed. Hence, as β-glucans are derived from D-glucose, the ratio of mannose to glucose in the cell wall was examined (Fig. 2A). Mannan is located in the outermost layer of the cell wall and covers β-glucans; therefore, it is believed that the ratio of mannose to glucose represents the degree of β-glucan exposure to the cell surface. In addition, the ratios of mannose to glucose in anp1Δ and gup1Δ, which are expected to have a low mannan content, were also measured. Anp1 is a subunit of the α-1,6-mannosyltransferase complex, which is responsible for the synthesis and initial branching of the long α-1,6-linked backbone of the hypermannose structure (Jungmann, Rayner, & Munro, 1999). Gup1 is involved in remodeling the GPI anchors that are required for the attachment of several CWPs to β-glucans (Bosson, Jaquenoud, & Conzelmann, 2006). As expected, the ratio of mannose to glucose in mcd4Δ was extremely low (< 0.1). The ratios in anp1Δ and gup1Δ were also remarkably reduced but were higher than that in mcd4Δ with an order of magnitude, as follows: mcd4Δ < anp1Δ < gup1Δ < WT. The macrophage activation ability was evaluated by measuring the amount of secreted TNF-α (Fig. 2B). The activity of mcd4Δ was again high, but the abilities of anp1Δ and gup1Δ were different. The activity of anp1Δ was as high as that of mcd4Δ, but the activity of gup1Δ was low, which might have been a result of low exposure of β-glucans, as
3. Results and discussion 3.1. Emulsion formation by mcd4Δ cells In order to analyze the degree of hydrophobicity on the mcd4Δ cell surface, emulsion formation from a previously obtained macrophage activation strain was observed when an mcd4Δ cell suspension was mixed with tetradecane. Emulsion formation was maintained for approximately 24 h, after which it weakened after 48 h (Fig. 1A). An equivalent emulsion was observed when kerosene was added instead of tetradecane. Such phenomena were not observed in WT cells. In the microscopic observation of the emulsion, yeast cells were dispersed, did not surround the micelles (Fig. 1B), and were not directly involved in micelle formation. Emulsion formation by yeast cells and oil has previously been reported (Meirelles et al., 2018); however, in that report, the amount of yeast used was greater than that used in this study, and considerable yeast precipitation was observed in the emulsifying test. In addition, there have been no reports on changes in the emulsification ability associated with a genetic mutation. 3
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Fig. 3. Emulsion formation by mixing kerosene with a cell suspension of Nlinked glycan mutants (A) and substances released from WT cells by Zymolyase100 T treatment (B).
defect makes it impossible to form N-linked glycans (Orlean, 2012), the effect of the deletion was examined. High activity of och1Δ, similar to that of mcd4Δ, was found; therefore, it was suggested that high-mannose N-linked glycans are not directly involved in emulsion formation, but the decrease in mannan from a defect in N-linked glycans induces this formation. Glycans might play a role in physically suppressing the release of CWPs involved in emulsion formation by cells.
Fig. 2. Ratio of mannose to glucose (A), macrophage activation (B), and emulsion formation (C) in anp1Δ, gup1Δ, and mcd4Δ. mcd4Δ cells cultured for 48 h and strains other than mcd4Δ cultured for 24 h were used.
observed in the different ratios of mannose to glucose. Finally, the emulsification abilities of the cells were evaluated (Fig. 2C), and it was observed that anp1Δ, but not gup1Δ, could form emulsions. In gup1Δ, some of the GPI anchor proteins leaked into the medium (Bosson et al., 2006); these leaked proteins might have an emulsifying activity. In contrast, anp1Δ showed activity similar to that of mcd4Δ, except that mcd4Δ was always stable and showed high activity, whereas anp1Δ occasionally showed weak activity under the same culture conditions, depending on the culture lot, suggesting that the activity of anp1Δ might be sensitive to differences in subtle culture conditions. There have been no reports on the relationship between the emulsification ability and genetic variation, but the results of mcd4Δ and anp1Δ evaluations suggested that there are other strains with a high emulsification ability.
3.4. Emulsifying activity of CWPs released from cells A soluble substance responsible for emulsion formation was reported to be released when the S. cerevisiae cell wall is hydrolyzed with β-1,3-glucanase (Cameron et al., 1988). The emulsifying activity of the substances was examined by treatment with β-1,3-glucanase. As shown in Fig. 3B, the released substances in the supernatant exhibited strong emulsion formation; therefore, the emulsification ability of mcd4Δ cells results from the release of proteins with an emulsification ability from the cell wall. CWPs are localized to the outermost layer of the cell in several ways; and in addition to binding using GPI anchors, there are noncovalently bound proteins (López-Ribot & Chaffin, 1996; Delgado et al., 2001). Most CWPs are released from the cells by the above-mentioned β-1,3glucanase treatment; however, it was presumed that it is difficult to release covalently bound proteins from cells mixed with oil. Therefore, the noncovalently bound proteins might be important for emulsion formation, and the emulsification ability of proteins easily released from the cell wall by washing the cells was subsequently examined. The emulsifying activity of the centrifuged supernatants containing proteins released after washing mcd4Δ cells cultured in a YPDS liquid medium was measured. When mcd4Δ cells were washed with PBSS, the supernatant created by centrifugation showed no emulsifying activity; however, when the cells of the pellet were resuspended in PBS and centrifuged, the supernatant induced emulsion formation (Fig. 4A). Morphological microscopic observations did not reveal any cell rupture when the cells were resuspended in PBS. Although emulsion formation induced by the supernatant was observed in och1Δ, it was not observed
3.3. Effect of N-linked glycans on the emulsification ability of cells Results indicated that high-mannose N-linked glycans are involved in the ability of cells to form emulsions. This effect was examined by observing emulsion formation by several mutants with a defect in Nlinked glycans. As Mnn10, Mnn11, Hoc1, and Anp1 are subunits of the α-1,6-mannosyltransferase complex (Orlean, 2012), the effect of these deletions was examined. Results showed that emulsion formation, despite being weak, was detected in mnn10Δ, mnn11Δ, and hoc1Δ (Fig. 3A); therefore, the fact that a decrease in mannan resulting from an abnormality in the α-1,6-mannosyltransferase complex enhances the emulsification ability of the cells could be confirmed. Next, as Och1 is involved in the first stages of N-linked glycan formation (i.e., the stage before the action of the α-1,6-mannosyltransferase complex) and the 4
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Fig. 4. Emulsion formation by mixing kerosene with substances released by washing cells with PBS. (A) Emulsion by the supernatant and pellet suspension after washing and (B) emulsion by substances after filtering the mcd4Δ supernatant using Amicon® Ultra Centrifugal Filters. Fig. 5. Emulsion criteria for screening and emulsion formation by substances released after washing the cells of the selected strains with PBS. (A) Criterion for cell suspensions, (B) criterion for substances released from cells, and (C) emulsion formed by substances released from cells.
in similarly prepared supernatants of WT cells; therefore, an emulsion might have been formed by proteins weakly bound to the cell wall, which were released from mutant cells in response to changes in the osmotic pressure when the cells were suspended in PBS. In order to estimate the molecular weight of the substance released from mcd4Δ cells that induce emulsion formation, the cell supernatants after resuspension in PBS were filtered using Amicon® Ultra Centrifugal Filters. Strong emulsifying activity was observed in the fractions that did not pass through the 10,000 MWCO filter and in those that did pass through the 100,000 MWCO filter (Fig. 4B); therefore, it could be confirmed that the substances responsible for inducing emulsion formation are not low-molecular-weight materials but have a high molecular weight in the order of tens of thousands. These proteins should be identified in future studies.
cell wall has been described previously (García et al., 2015). Asc1 is a core component of the small (40S) ribosomal subunit, G-protein βsubunit, and guanine dissociation inhibitor for Gpa2p. The direct involvement of Asc1 in the cell wall is also not known; however, the disruption of Asc1 results in increased chitin deposition within the cell wall (Lesage et al., 2005). Therefore, asc1Δ and scp160Δ are expected to produce changes in the cell wall structure. 3.6. Characterization of asc1Δ and scp160Δ Macrophage activation of asc1Δ and scp160Δ in MATa was evaluated by measuring the amount of secreted TNF-α (Fig. 6A). Although the activity of scp160Δ with strong emulsification ability was statistically increased compared to that of WT (P < 0.05), the macrophage activation abilities were not as strong as that of mcd4Δ (Fig. 2B). There was no statistically significant difference between asc1Δ and WT cells. Furthermore, the ratio of mannose to glucose was examined (Fig. 6B). asc1Δ and scp160Δ in MATa showed no remarkable decrease in the ratio of mannose to glucose compared to that of mcd4Δ and anp1Δ (Fig. 2A). Thus, a large decrease in mannan is necessary for a strong macrophage activation ability; however, it is suggested that there are also other factors involved in the emulsification ability.
3.5. Screening yeast strains with high emulsifying activity Strains with significantly reduced mannan have a strong ability to form emulsions. In order to examine other factors that induce emulsion formation, the Yeast Knockout deletion collection (5154 strains) constructed from the BY4741 parental strain (MATa) was screened. Because measuring cell emulsion formation is very simple, the emulsifying activity of the cells was used for this screening. To simplify the experiment, cells growing on a YPDS solid medium were picked up, and emulsion formation was evaluated using an Eppendorf tube, as shown in Fig. 5A. About 5076 strains showed no activity (evaluated as −). Strains that showed an emulsion similar to that of mcd4Δ (evaluated as ++) were selected, and the reproducibility of the effect of those gene deletions was confirmed using strains of MATα lacking the same gene. In the screening, eight strains (arp1Δ, asc1Δ, scp160Δ, pop2Δ, clc1Δ, hpr1Δ, ymr001c-aΔ, and rvs161Δ) whose emulsification activity was categorized as ++ in both MATa and MATα were selected. Next, the emulsifying activity of centrifuged supernatants of the cells resuspended in PBS was measured using the selected strains. The criteria for the evaluation are shown in Fig. 5B. Finally, asc1Δ and scp160Δ, which showed strong emulsifying activity equivalent to that of mcd4Δ in both MATa and MATα, were selected (Fig. 5C). Scp160 is an RNA-binding protein, and Scp160-dependent mRNA trafficking is known to be involved in pheromone-gradient sensing and chemotropism (Gelin-Licht, Paliwal, Conlon, Levchenko, & Gerst, 2012). Scp160 is not known to be directly involved in cell wall formation; however, the relationship between RNA metabolism by Scp160 and the
4. Conclusion In mcd4Δ, which has a defect in the cell wall, the cells themselves, or the substances released by washing the cells with PBS, have strong emulsifying activity. As emulsification is a commonly used process in food production and S. cerevisiae is a safe and convenient microorganism used in food fermentation, a yeast-derived emulsifier might be very useful in the industry. This study also showed that strains that have a defect in the formation of N-linked glycans (anp1Δ, och1Δ, mnn10Δ, mnn11Δ, and hoc1Δ) have an emulsification ability. Furthermore, after screening the strains that have emulsifying activity, two strains (asc1Δ and scp160Δ) with a strong emulsification ability without a large decrease in the mannan content were found. Although the direct effect of these defective genes on cell wall synthesis is unknown, they do affect the cell wall structure, and cell wall changes are considered to be important factors that influence the emulsifying 5
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Fig. 6. Macrophage activation by contact with yeast cells (A) and the ratio of mannose to glucose (B) in asc1Δ and scp160Δ. development: Yeast-derived β-glucan particles. Human Vaccines & Immunotherapeutics, 10, 1309–1318. Delgado, M. L., O'Connor, J. E., Azorín, I., Renau-Piqueras, J., Gil, M. L., & Gozalbo, D. (2001). The glyceraldehyde-3-phosphate dehydrogenase polypeptides encoded by the Saccharomyces cerevisiae TDH1, TDH2 and TDH3 genes are also cell wall proteins. Microbiology, 147(Pt 2), 411–417. Dikit, P., Maneerat, S., Musikasang, H., & H-kittikun, A. (2010). Emulsifier properties of the mannoprotein extract from yeast isolated from sugar palm wine. ScienceAsia, 36, 312–318. Gelin-Licht, R., Paliwal, S., Conlon, P., Levchenko, A., & Gerst, J. E. (2012). Scp160dependent mRNA trafficking mediates pheromone gradient sensing and chemotropism in yeast. Cell Reports, 1, 483–494. García, R., Botet, J., Rodríguez-Peña, J. M., Bermejo, C., Ribas, J. C., Revuelta, J. L., et al. (2015). Genomic profiling of fungal cell wall-interfering compounds: Identification of a common gene signature. BMC Genomics, 16, 683. Ikeda, Y., Adachi, Y., Ishii, T., Miura, N., Tamura, H., & Ohno, N. (2008). Dissociation of Toll-like receptor 2-mediated innate immune response to Zymosan by organic solvent-treatment without loss of Dectin-1 reactivity. Biological &/and Pharmaceutical Bulletin, 31, 13–18. Jungmann, J., Rayner, J. C., & Munro, S. (1999). The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a Golgi mannosyltransferase complex. Journal of Biological Chemistry, 274, 6579–6585. Klis, F. M., Boorsma, A., & De Groot, P. W. (2006). Cell wall construction in Saccharomyces cerevisiae. Yeast, 23, 185–202. Lesage, G., Shapiro, J., Specht, C. A., Sdicu, A. M., Ménard, P., Hussein, S., et al. (2005). An interactional network of genes involved in chitin synthesis in Saccharomyces cerevisiae. BMC Genetics, 6, 8. Lesage, G., & Bussey, H. (2006). Cell wall assembly in Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews, 70, 317–343. Li, J., & Karboune, S. (2019). Characterization of the composition and the techno-functional properties of mannoproteins from Saccharomyces cerevisiae yeast cell walls. Food Chemistry, 297, 124867. https://doi.org/10.1016/j.foodchem.2019.05.141. López-Ribot, J. L., & Chaffin, W. L. (1996). Members of the Hsp70 family of proteins in the cell wall of Saccharomyces cerevisiae. Journal of Bacteriology, 178, 4724–4726. Maneesri, J., Azuma, M., Sakai, Y., Igarashi, K., Matsumoto, T., Fukuda, H., et al. (2005). Deletion of MCD4 involved in glycosylphosphatidylinositol (GPI) anchor synthesis leads to an increase in β-1,6-glucan level and a decrease in GPI-anchored protein and mannan levels in the cell wall of Saccharomyces cerevisiae. Journal of Bioscience and Bioengineering, 99, 354–360. Meirelles, A. A. D., da Cunha, R. L., & Gombert, A. K. (2018). The role of Saccharomyces cerevisiae in stabilizing emulsions of hexadecane in aqueous media. Applied Microbiology and Biotechnology, 102, 3411–3424. Novak, M., & Vetvicka, V. (2008). β-Glucans, history, and the present: Immunomodulatory aspects and mechanisms of action. Journal of Immunotoxicology, 5, 47–57. Orlean, P. (2012). Architecture and biosynthesis of the Saccharomyces cerevisiae cell wall. Genetics, 192, 775–818. Sakai, Y., Azuma, M., Takada, Y., Umeyama, T., Kaneko, A., Fujita, T., et al. (2007). Saccharomyces cerevisiae mutant displaying β-glucans on cell surface. Journal of Bioscience and Bioengineering, 103, 161–166. Takada, Y., Nishino, Y., Ito, C., Watanabe, H., Kanzaki, K., Tachibana, T., et al. (2014). Isolation and characterization of baker's yeast capable of strongly activating a macrophage. FEMS Yeast Research, 14, 261–269. Takada, Y., Mizobuchi, A., Kato, T., Kasahara, E., Ito, C., Watanabe, H., et al. (2014). Isolation of diploid baker's yeast capable of strongly activating immune cells and analyses of the cell wall structure. Bioscience, Biotechnology, and Biochemistry, 78, 911–915. Winzeler, E. A., Shoemaker, D. D., Astromoff, A., Liang, H., Anderson, K., Andre, B., et al. (1999). Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science, 285, 901–906. Young, S. H., Ye, J., Frazer, D. G., Shi, X., & Castranova, V. (2001). Molecular mechanism of tumor necrosis factor-alpha production in 1→3-β-glucan (zymosan)-activated macrophages. Journal of Biological Chemistry, 276, 20781–20787.
activity. There have been no reports that such mutations affect emulsion formation, but the accumulation of such findings is essential for the development of a yeast-derived emulsifier. The findings from this study could be useful for this development. Construction of additional promising strains is expected by, for example, combining the mutations found in this research. CRediT authorship contribution statement Shinsuke Nerome: Data curation, Formal analysis, Writing - original draft, Writing - review & editing. Masaya Onishi: Data curation, Formal analysis. Daiki Saito: Data curation, Formal analysis. Ayano Mizobuchi: Data curation, Formal analysis. Tatsuya Ando: Data curation, Formal analysis. Yui Daira: Data curation, Formal analysis. Azusa Matsumoto: Data curation, Formal analysis. Yoshihiro Ojima: Project administration, Writing - review & editing. Masayuki Azuma: Funding acquisition, Project administration, Writing - original draft, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by funding from the Institute for Fermentation, Osaka, Japan and the Public Foundation of Elizabeth Arnold-Fuji, Japan. References Bosson, R., Jaquenoud, M., & Conzelmann, A. (2006). GUP1 of Saccharomyces cerevisiae encodes an O-acyltransferase involved in remodeling of the GPI anchor. Molecular Biology of the Cell, 17, 2636–2645. Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J. P., Hieter, & Boeke, J. D. (1998). Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast, 14, 115–132. Cabib, E., & Arroyo, J. (2013). How carbohydrates sculpt cells: Chemical control of morphogenesis in the yeast cell wall. Nature Reviews Microbiology, 11, 648–655. Cameron, D. R., Cooper, D. G., & Neufeld, R. J. (1988). The mannoprotein of Saccharomyces cerevisiae is an effective bioemulsifier. Applied and Environment Microbiology, 54, 1420–1425. Dallies, N., Francqois, J., & Paquet, V. (1998). A new method for quantitative determination of polysaccharides in the yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae. Yeast, 14, 1297–1306. De Iseppi, A., Curioni, A., Marangon, M., Vincenzi, S., Kantureeva, G., & Lomolino, G. (2019). Characterization and emulsifying properties of extracts obtained by physical and enzymatic methods from an oenological yeast strain. Journal of the Science of Food and Agriculture. https://doi.org/10.1002/jsfa.9833. De Smet, R., Allais, L., & Cuvelier, C. A. (2014). Recent advances in oral vaccine
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Cell surface changes that advance the application of using yeast as a food emulsifier
T
Shinsuke Nerome, Masaya Onishi, Daiki Saito, Ayano Mizobuchi, Tatsuya Ando, Yui Daira, ⁎ Azusa Matsumoto, Yoshihiro Ojima, Masayuki Azuma Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, Sugimoto 3-3-138, Sumiyoshi-ku, Osaka 558-8585, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Emulsification Yeast Cell wall Mannan Emulsifier
A previous study revealed that Saccharomyces cerevisiae mcd4Δ, a cell wall mutant with a defect in the synthesis of the glycosylphosphatidylinositol anchor, has a strong macrophage activation ability. In this study, remarkable emulsion formation after cell suspensions of mcd4Δ and anp1Δ (which exhibit an extreme reduction of mannan) were mixed with oil was found. Moreover, the relationship between cell wall mutation and emulsion formation was investigated, suggesting that och1Δ with a defect in the formation of N-linked glycans also had a strong emulsification ability and that high molecular weight materials released from the cells were involved in emulsion formation. Furthermore, two strains (asc1Δ and scp160Δ) with a strong emulsification ability without a large decrease in mannan content were also found from the wide screening of strains that exhibit an emulsifying activity using more than 5000 gene-deficient strains. These results provide valuable information for the development of a yeast-derived emulsifier.
1. Introduction Emulsification is a procedure frequently performed in the food and cosmetics industries in which emulsifiers are used. In general, chemically synthesized emulsifiers are often used; however, some naturally derived emulsifiers like casein are also used, but not in large quantities. Yeast is presumably a natural emulsifier. If yeast can have both fermentative and adequate emulsification abilities, there will be no need to add chemically synthesized emulsifiers to many food products that use yeast, such as bread. There are a few reports on the ability of yeast to form emulsions; for example, mannoproteins obtained from β-1,3glucanase or physical treatment of cells have been reported to have an emulsifying effect (Cameron, Cooper, & Neufeld, 1988; De Iseppi et al., 2019; Dikit, Maneerat, Musikasang, & H-kittikun, 2010; Li & Karboune, 2019). In addition, research has been conducted on the oil-in-water emulsion formed by Saccharomyces cerevisiae cells for use in oil production (Meirelles, da Cunha, & Gombert, 2018); however, so far, there is no research on how to enhance the emulsification ability of yeast. In order to use yeast as an emulsifier in food, it is necessary to overcome the cost barrier of preparation; developing yeast suitable for this use is one of the solutions.
The cell wall of S. cerevisiae comprises mannoproteins, β-1,3-glucan, β-1,6-glucan, and chitin, which account for approximately 30–50%, 30–45%, 5–10%, and 1.5–6%, respectively, of the dry cell wall weight (Klis, Boorsma, & De Groot, 2006). The majority of mannoproteins are covalently coupled to cell wall polysaccharides and are divided into two types: glycosylphosphatidylinositol cell wall proteins (GPI-CWPs) and alkali-sensitive linkage CWPs. GPI-CWPs are attached to β-glucans through GPI anchors. These proteins contain high-mannose N-linked glycans and are localized in the outermost layer of the cell wall, covering the cells with mannan (Klis et al., 2006; Lesage & Bussey, 2006; Orlean, 2012; Cabib & Arroyo, 2013). In addition, there are noncovalently bound CWPs, such as heat shock proteins and glycolytic enzymes (López-Ribot & Chaffin, 1996; Delgado et al., 2001), which are released from the cells by a reducing agent under weak alkaline conditions. These noncovalently bound proteins are trapped inside the cell wall or weakly ionically bound to other CWPs. The physiological functions of β-glucans are well known (Novak & Vetvicka, 2008), and the immunomodulatory effects of S. cerevisiae βglucans have been reported previously (Young, Ye, Frazer, Shi, & Castranova, 2001; Ikeda et al., 2008; De Smet, Allais, & Cuvelier, 2014). The immunomodulatory effects of whole S. cerevisiae cells have been
Abbreviations: GPI, glycosylphosphatidylinositol; CWP, cell wall protein; TNF-α, tumor necrosis factor-α; MATa, mating type a; MATα, mating type α; WT, wild type; PBS, phosphate-buffered saline; YPD, yeast extract peptone dextrose; PBSS, phosphate-buffered saline containing sorbitol; MWCO, molecular weight cutoff ⁎ Corresponding author. E-mail address: [email protected] (M. Azuma). https://doi.org/10.1016/j.foodchem.2020.126264 Received 3 October 2019; Received in revised form 10 January 2020; Accepted 17 January 2020 Available online 21 January 2020 0308-8146/ © 2020 Elsevier Ltd. All rights reserved.
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2.4. Analyses of substances released from yeast cells
the focus because of their beneficial effects on host immunity through fermented foods containing yeast (Sakai et al., 2007; Takada, Nishino et al., 2014, Takada, Mizobuchi et al., 2014). Previous studies have reported a large decrease in cell wall mannan in the mutant strain mcd4Δ, which is defective in the synthesis of GPI anchors necessary for fixing mannoproteins to β-glucans (Maneesri et al., 2005). These mutant mcd4Δ cells strongly activated mouse macrophages through direct contact in vitro and induced high secretion of tumor necrosis factor-α (TNF-α). Mouse immune cells were also activated in vivo by contact with mcd4Δ cells (Sakai et al., 2007). In this study, the analysis of mcd4Δ processes showed remarkable emulsion formation when cell suspensions were mixed with oil. With this result as a trigger, focus was placed on gene mutations that enhance the emulsification ability. The effects of various genetic mutations were examined, and cell surface changes that lead to the high emulsifying activity of cells were found. In addition, it was confirmed that CWPs easily released from yeast cells are involved in emulsion formation.
The OD600 value of mcd4Δ cells after cultivation for 48 h was adjusted to 15 using PBSS, and a 20 mL cell suspension was centrifuged at 3000×g for 5 min, after which the pellet was resuspended in 20 mL of PBS. The suspension was then vortexed and centrifuged at 3000×g for 5 min. The supernatant (10 mL) was filtered through an Amicon® Ultra Centrifugal Filter (10,000 or 100,000 molecular weight cutoff, MWCO), and any residual material that did not pass through the filter was diluted to its original volume using PBS. The emulsifying activities of the unfiltered material (top chamber) and the filtrate (bottom chamber) were measured as described above. 2.5. Emulsion formation by substances released from yeast cells after treatment with β-1,3-glucanase Wild type (WT) cells were cultured in a YPD medium at 30 ℃ overnight, harvested by centrifugation, and washed with 66 mM PBS (pH 7.5) containing 1.2 M sorbitol. The OD600 value was adjusted to either 1.0 or 3.0 with the buffer. β-1,3-Glucanase (Zymolyase-100T) was added to the cell suspension (4 mL) to 10 units/mL of the final concentration. The suspension was then incubated at 35℃ for 2 h and centrifuged at 700×g for 5 min. The pellet was washed with the buffer and then resuspended in 4 mL of buffer. The supernatant or pellet suspension (2 mL) was mixed with 1 mL of kerosene, and emulsion formation was assessed.
2. Materials and methods 2.1. Strains and media S. cerevisiae BY4741 (mating type a [MATa] his3Δ1 leu2 Δ 0 met15Δ0 ura3 Δ 0) was used as the parental strain (Brachmann et al., 1998), and each knockout strain (BY4741, orf Δ::kanMX4) (Winzeler et al., 1999) was purchased from GE Healthcare UK Ltd. (Buckinghamshire, UK) and used for screening. In this paper, for example, mcd4 gene-deficient strain is denoted as mcd4Δ. For mcd4Δ (MATa) only, the strain that was obtained previously from the heterozygous strain was used (Maneesri et al., 2005). S. cerevisiae BY4742 (mating type α [MATα] his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) and each knockout strain (BY4742, orf Δ::kanMX4) was used to confirm the effects of gene deletions selected in the screening. A yeast extract peptone dextrose (YPD) medium (1% [w/v] yeast extract, 2% [w/v] peptone, and 2% [w/v] glucose) was used to culture the yeast strains. The yeast extract was purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). Sorbitol (0.6 M) was added to the YPD medium when osmotic support was necessary; this medium was denoted as YPDS.
2.6. Screening of yeast strains with emulsifying activity First, deletion strains (MATa) of the Yeast Knockout deletion collection were screened. In order to make the operation as easy as possible, the cells were cultured on YPDS agar at 30 ℃ for 48 h, and approximately equal amounts were visibly judged and gathered using a toothpick from single colonies on the plates and suspended in PBS. A 0.5 mL suspension was mixed with 0.5 mL of kerosene in a 1.5 mL Eppendorf tube and incubated at 30 ℃ for 1 h to observe emulsion formation. Emulsification similar to that of mcd4Δ was evaluated as + +, and the emulsification ability of the tested strains was divided into four categories ranging from −to ++. Strains that exhibited ++ emulsification ability were selected, and the effects of those deletion genes were confirmed from the emulsification ability of gene deletion strains of MATα. In order to further investigate the details of emulsion formation with the eight selected strains, the cells were cultured at 30 ℃ for 48 h and harvested by centrifugation at 3000×g for 5 min, and the OD600 value was adjusted to 0.5 with PBSS. A YPDS liquid medium was used for the culture. The cell suspension (50 mL) was centrifuged, and the pellet was resuspended in 7 mL of PBS. The cells were then centrifuged again, and the supernatant was collected and lyophilized. The lyophilized material was resuspended in 1.1 mL of pure water; a 1 mL sample was mixed with 1 mL of kerosene, and emulsion formation was observed. Emulsification similar to that of mcdΔ was assessed as +++, and the emulsification ability of mutants was divided into five categories ranging from − to +++. Mutants that showed an emulsification ability of +++ in both MATa and MATα were selected.
2.2. Emulsion formation by yeast cells Each strain was cultured in a YPDS liquid medium at 30 ℃ for 48 h, and the cells were harvested by centrifugation at 3000×g for 5 min. In order to measure emulsion formation by yeast cells, the optical density at 600 nm (OD600) was adjusted to 0.7 using phosphate-buffered saline (PBS) containing 0.6 M sorbitol (PBSS), and a 5 mL cell suspension was centrifuged at 3000×g for 5 min. The pellet was then resuspended in 5 mL of PBS, and 1 mL of either kerosene or tetradecane was added. After vortexing for 30 s, the suspension was incubated at 30 ℃ for 1, 24, or 48 h and photographed after each time period. The image of micelles immediately after mixing tetradecane with the mcd4Δ cell suspension cultured for 48 h was observed under an Olympus BH50 microscope (Olympus, Tokyo, Japan). 2.3. Emulsion formation by substances released from yeast cells
2.7. Macrophage activation by contact with yeast cells Each strain was cultured in a YPDS liquid medium at 30 ℃ for 48 h. After harvesting, the OD600 value was adjusted to 0.7 using PBSS. The cell suspension (5 mL) was then centrifuged at 3000×g for 5 min, and the pellet was resuspended in 5 mL of PBS and centrifuged again at 3000×g for 5 min. The supernatant (5 mL) and 1 mL of kerosene were mixed, and emulsion formation was assessed as described above. After the last centrifugation, the precipitate was resuspended in 5 mL of PBS and mixed with 1 mL of kerosene, after which emulsion formation was assessed.
Macrophage activation by contact with yeast cells was measured according to a previously described method with some modifications (Sakai et al., 2007). Yeast cells cultured at 30 ℃ for 24 h in a YPDS medium were harvested and washed twice with PBSS. mcd4Δ cells were cultured for 48 h because of their slow growth. The cells were then fixed in 70% (v/v) ethanol and lyophilized. The dried cells were resuspended in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (ICN 2
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Biomedicals, Aurora, OH, USA). The mouse macrophage line RAW264.7 (ATCC TIB-71) was also precultured in RPMI-1640, washed twice with medium, counted, and resuspended in fresh medium at 0.5 × 106 cells/mL. The cells were then cultured for 24 h, the culture medium was removed, and 0.5 mL of new medium containing yeast cells at a final concentration of 100 μg/mL was added. After adding the yeast samples to the macrophages, the solution was incubated in 5% CO2 at 37 ℃ for 6 h and centrifuged, and the supernatant was recovered. The levels of mouse TNF-α in the supernatants were measured using enzyme-linked immunosorbent assay according to the manufacturer’s instructions (R&D Systems, Inc., Minneapolis, MN, USA). In addition, baker’s yeast-derived insoluble β-glucan (Oriental Yeast Co., Ltd.) was used as the control. The final concentration was 50 μg/mL. 2.8. Ratio of mannose to glucose in the cell wall The amounts of glucose and mannose in the cell wall were evaluated according to a previously reported method (Dallies, Francqois, & Paquet, 1998; Takada, Nishino et al., 2014). The cells were cultured at 30 ℃ for 24 h, harvested by centrifugation at 3000×g for 5 min, and washed twice with PBSS. mcd4Δ cells were cultured for 48 h in a YPDS liquid medium. The cells were then broken using glass beads on ice, and cell walls were collected by centrifugation at 3800×g for 5 min and then freeze-dried. Then, 3 mg of the collected cell walls was suspended in 75 μL of 72% (w/w) H2SO4 and kept at room temperature for 3 h. The obtained slurry was diluted to 1 mL with pure water and heated at 100 ℃ for 4 h. The suspension was then cooled on ice and neutralized with saturated Ba(OH)2. The supernatant was collected by centrifugation at 3800×g for 5 min and kept at 4 ℃ overnight. The precipitate was removed by centrifugation, and the supernatant was freeze-dried. The amounts of glucose and mannose were measured using a highperformance liquid chromatograph equipped with an RID-6A detector (Shimadzu Corp., Kyoto, Japan) and a Rezex RPM-Monosaccharide column (300 × 7 mm; Phenomenex, Torrance, CA, USA). Milli-Q water (Millipore Corp., Bedford, MA, USA) was used for the elution. The flow rate and column temperature were 1.0 mL/min and 78 ℃, respectively.
Fig. 1. Emulsion formation by mixing oil with an mcd4Δ cell suspension cultured for 24 h (A) and a microscopic image of micelles formed by mixing kerosene and an mcd4Δ cell suspension (B).
2.9. Statistical analysis In order to measure the ratio of mannose to glucose in the cell wall as well as macrophage activation by contact with yeast cells, all results are shown as the mean ± standard deviation of independent experiments (n ≥ 3). Welch’s t-test was used to compare two groups. Differences were assessed using a two-sided test with an ⍺-level of 0.05. Significant differences from WT were indicated by an asterisk (*P < 0.05).
3.2. Emulsion formation by anp1Δ cells Mannan staining using Con A-FITC showed that the amount of mannan in the mcd4Δ cell wall was extremely low (Maneesri et al., 2005); however, no quantitative evaluation was performed. Hence, as β-glucans are derived from D-glucose, the ratio of mannose to glucose in the cell wall was examined (Fig. 2A). Mannan is located in the outermost layer of the cell wall and covers β-glucans; therefore, it is believed that the ratio of mannose to glucose represents the degree of β-glucan exposure to the cell surface. In addition, the ratios of mannose to glucose in anp1Δ and gup1Δ, which are expected to have a low mannan content, were also measured. Anp1 is a subunit of the α-1,6-mannosyltransferase complex, which is responsible for the synthesis and initial branching of the long α-1,6-linked backbone of the hypermannose structure (Jungmann, Rayner, & Munro, 1999). Gup1 is involved in remodeling the GPI anchors that are required for the attachment of several CWPs to β-glucans (Bosson, Jaquenoud, & Conzelmann, 2006). As expected, the ratio of mannose to glucose in mcd4Δ was extremely low (< 0.1). The ratios in anp1Δ and gup1Δ were also remarkably reduced but were higher than that in mcd4Δ with an order of magnitude, as follows: mcd4Δ < anp1Δ < gup1Δ < WT. The macrophage activation ability was evaluated by measuring the amount of secreted TNF-α (Fig. 2B). The activity of mcd4Δ was again high, but the abilities of anp1Δ and gup1Δ were different. The activity of anp1Δ was as high as that of mcd4Δ, but the activity of gup1Δ was low, which might have been a result of low exposure of β-glucans, as
3. Results and discussion 3.1. Emulsion formation by mcd4Δ cells In order to analyze the degree of hydrophobicity on the mcd4Δ cell surface, emulsion formation from a previously obtained macrophage activation strain was observed when an mcd4Δ cell suspension was mixed with tetradecane. Emulsion formation was maintained for approximately 24 h, after which it weakened after 48 h (Fig. 1A). An equivalent emulsion was observed when kerosene was added instead of tetradecane. Such phenomena were not observed in WT cells. In the microscopic observation of the emulsion, yeast cells were dispersed, did not surround the micelles (Fig. 1B), and were not directly involved in micelle formation. Emulsion formation by yeast cells and oil has previously been reported (Meirelles et al., 2018); however, in that report, the amount of yeast used was greater than that used in this study, and considerable yeast precipitation was observed in the emulsifying test. In addition, there have been no reports on changes in the emulsification ability associated with a genetic mutation. 3
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Fig. 3. Emulsion formation by mixing kerosene with a cell suspension of Nlinked glycan mutants (A) and substances released from WT cells by Zymolyase100 T treatment (B).
defect makes it impossible to form N-linked glycans (Orlean, 2012), the effect of the deletion was examined. High activity of och1Δ, similar to that of mcd4Δ, was found; therefore, it was suggested that high-mannose N-linked glycans are not directly involved in emulsion formation, but the decrease in mannan from a defect in N-linked glycans induces this formation. Glycans might play a role in physically suppressing the release of CWPs involved in emulsion formation by cells.
Fig. 2. Ratio of mannose to glucose (A), macrophage activation (B), and emulsion formation (C) in anp1Δ, gup1Δ, and mcd4Δ. mcd4Δ cells cultured for 48 h and strains other than mcd4Δ cultured for 24 h were used.
observed in the different ratios of mannose to glucose. Finally, the emulsification abilities of the cells were evaluated (Fig. 2C), and it was observed that anp1Δ, but not gup1Δ, could form emulsions. In gup1Δ, some of the GPI anchor proteins leaked into the medium (Bosson et al., 2006); these leaked proteins might have an emulsifying activity. In contrast, anp1Δ showed activity similar to that of mcd4Δ, except that mcd4Δ was always stable and showed high activity, whereas anp1Δ occasionally showed weak activity under the same culture conditions, depending on the culture lot, suggesting that the activity of anp1Δ might be sensitive to differences in subtle culture conditions. There have been no reports on the relationship between the emulsification ability and genetic variation, but the results of mcd4Δ and anp1Δ evaluations suggested that there are other strains with a high emulsification ability.
3.4. Emulsifying activity of CWPs released from cells A soluble substance responsible for emulsion formation was reported to be released when the S. cerevisiae cell wall is hydrolyzed with β-1,3-glucanase (Cameron et al., 1988). The emulsifying activity of the substances was examined by treatment with β-1,3-glucanase. As shown in Fig. 3B, the released substances in the supernatant exhibited strong emulsion formation; therefore, the emulsification ability of mcd4Δ cells results from the release of proteins with an emulsification ability from the cell wall. CWPs are localized to the outermost layer of the cell in several ways; and in addition to binding using GPI anchors, there are noncovalently bound proteins (López-Ribot & Chaffin, 1996; Delgado et al., 2001). Most CWPs are released from the cells by the above-mentioned β-1,3glucanase treatment; however, it was presumed that it is difficult to release covalently bound proteins from cells mixed with oil. Therefore, the noncovalently bound proteins might be important for emulsion formation, and the emulsification ability of proteins easily released from the cell wall by washing the cells was subsequently examined. The emulsifying activity of the centrifuged supernatants containing proteins released after washing mcd4Δ cells cultured in a YPDS liquid medium was measured. When mcd4Δ cells were washed with PBSS, the supernatant created by centrifugation showed no emulsifying activity; however, when the cells of the pellet were resuspended in PBS and centrifuged, the supernatant induced emulsion formation (Fig. 4A). Morphological microscopic observations did not reveal any cell rupture when the cells were resuspended in PBS. Although emulsion formation induced by the supernatant was observed in och1Δ, it was not observed
3.3. Effect of N-linked glycans on the emulsification ability of cells Results indicated that high-mannose N-linked glycans are involved in the ability of cells to form emulsions. This effect was examined by observing emulsion formation by several mutants with a defect in Nlinked glycans. As Mnn10, Mnn11, Hoc1, and Anp1 are subunits of the α-1,6-mannosyltransferase complex (Orlean, 2012), the effect of these deletions was examined. Results showed that emulsion formation, despite being weak, was detected in mnn10Δ, mnn11Δ, and hoc1Δ (Fig. 3A); therefore, the fact that a decrease in mannan resulting from an abnormality in the α-1,6-mannosyltransferase complex enhances the emulsification ability of the cells could be confirmed. Next, as Och1 is involved in the first stages of N-linked glycan formation (i.e., the stage before the action of the α-1,6-mannosyltransferase complex) and the 4
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Fig. 4. Emulsion formation by mixing kerosene with substances released by washing cells with PBS. (A) Emulsion by the supernatant and pellet suspension after washing and (B) emulsion by substances after filtering the mcd4Δ supernatant using Amicon® Ultra Centrifugal Filters. Fig. 5. Emulsion criteria for screening and emulsion formation by substances released after washing the cells of the selected strains with PBS. (A) Criterion for cell suspensions, (B) criterion for substances released from cells, and (C) emulsion formed by substances released from cells.
in similarly prepared supernatants of WT cells; therefore, an emulsion might have been formed by proteins weakly bound to the cell wall, which were released from mutant cells in response to changes in the osmotic pressure when the cells were suspended in PBS. In order to estimate the molecular weight of the substance released from mcd4Δ cells that induce emulsion formation, the cell supernatants after resuspension in PBS were filtered using Amicon® Ultra Centrifugal Filters. Strong emulsifying activity was observed in the fractions that did not pass through the 10,000 MWCO filter and in those that did pass through the 100,000 MWCO filter (Fig. 4B); therefore, it could be confirmed that the substances responsible for inducing emulsion formation are not low-molecular-weight materials but have a high molecular weight in the order of tens of thousands. These proteins should be identified in future studies.
cell wall has been described previously (García et al., 2015). Asc1 is a core component of the small (40S) ribosomal subunit, G-protein βsubunit, and guanine dissociation inhibitor for Gpa2p. The direct involvement of Asc1 in the cell wall is also not known; however, the disruption of Asc1 results in increased chitin deposition within the cell wall (Lesage et al., 2005). Therefore, asc1Δ and scp160Δ are expected to produce changes in the cell wall structure. 3.6. Characterization of asc1Δ and scp160Δ Macrophage activation of asc1Δ and scp160Δ in MATa was evaluated by measuring the amount of secreted TNF-α (Fig. 6A). Although the activity of scp160Δ with strong emulsification ability was statistically increased compared to that of WT (P < 0.05), the macrophage activation abilities were not as strong as that of mcd4Δ (Fig. 2B). There was no statistically significant difference between asc1Δ and WT cells. Furthermore, the ratio of mannose to glucose was examined (Fig. 6B). asc1Δ and scp160Δ in MATa showed no remarkable decrease in the ratio of mannose to glucose compared to that of mcd4Δ and anp1Δ (Fig. 2A). Thus, a large decrease in mannan is necessary for a strong macrophage activation ability; however, it is suggested that there are also other factors involved in the emulsification ability.
3.5. Screening yeast strains with high emulsifying activity Strains with significantly reduced mannan have a strong ability to form emulsions. In order to examine other factors that induce emulsion formation, the Yeast Knockout deletion collection (5154 strains) constructed from the BY4741 parental strain (MATa) was screened. Because measuring cell emulsion formation is very simple, the emulsifying activity of the cells was used for this screening. To simplify the experiment, cells growing on a YPDS solid medium were picked up, and emulsion formation was evaluated using an Eppendorf tube, as shown in Fig. 5A. About 5076 strains showed no activity (evaluated as −). Strains that showed an emulsion similar to that of mcd4Δ (evaluated as ++) were selected, and the reproducibility of the effect of those gene deletions was confirmed using strains of MATα lacking the same gene. In the screening, eight strains (arp1Δ, asc1Δ, scp160Δ, pop2Δ, clc1Δ, hpr1Δ, ymr001c-aΔ, and rvs161Δ) whose emulsification activity was categorized as ++ in both MATa and MATα were selected. Next, the emulsifying activity of centrifuged supernatants of the cells resuspended in PBS was measured using the selected strains. The criteria for the evaluation are shown in Fig. 5B. Finally, asc1Δ and scp160Δ, which showed strong emulsifying activity equivalent to that of mcd4Δ in both MATa and MATα, were selected (Fig. 5C). Scp160 is an RNA-binding protein, and Scp160-dependent mRNA trafficking is known to be involved in pheromone-gradient sensing and chemotropism (Gelin-Licht, Paliwal, Conlon, Levchenko, & Gerst, 2012). Scp160 is not known to be directly involved in cell wall formation; however, the relationship between RNA metabolism by Scp160 and the
4. Conclusion In mcd4Δ, which has a defect in the cell wall, the cells themselves, or the substances released by washing the cells with PBS, have strong emulsifying activity. As emulsification is a commonly used process in food production and S. cerevisiae is a safe and convenient microorganism used in food fermentation, a yeast-derived emulsifier might be very useful in the industry. This study also showed that strains that have a defect in the formation of N-linked glycans (anp1Δ, och1Δ, mnn10Δ, mnn11Δ, and hoc1Δ) have an emulsification ability. Furthermore, after screening the strains that have emulsifying activity, two strains (asc1Δ and scp160Δ) with a strong emulsification ability without a large decrease in the mannan content were found. Although the direct effect of these defective genes on cell wall synthesis is unknown, they do affect the cell wall structure, and cell wall changes are considered to be important factors that influence the emulsifying 5
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Fig. 6. Macrophage activation by contact with yeast cells (A) and the ratio of mannose to glucose (B) in asc1Δ and scp160Δ. development: Yeast-derived β-glucan particles. Human Vaccines & Immunotherapeutics, 10, 1309–1318. Delgado, M. L., O'Connor, J. E., Azorín, I., Renau-Piqueras, J., Gil, M. L., & Gozalbo, D. (2001). The glyceraldehyde-3-phosphate dehydrogenase polypeptides encoded by the Saccharomyces cerevisiae TDH1, TDH2 and TDH3 genes are also cell wall proteins. Microbiology, 147(Pt 2), 411–417. Dikit, P., Maneerat, S., Musikasang, H., & H-kittikun, A. (2010). Emulsifier properties of the mannoprotein extract from yeast isolated from sugar palm wine. ScienceAsia, 36, 312–318. Gelin-Licht, R., Paliwal, S., Conlon, P., Levchenko, A., & Gerst, J. E. (2012). Scp160dependent mRNA trafficking mediates pheromone gradient sensing and chemotropism in yeast. Cell Reports, 1, 483–494. García, R., Botet, J., Rodríguez-Peña, J. M., Bermejo, C., Ribas, J. C., Revuelta, J. L., et al. (2015). Genomic profiling of fungal cell wall-interfering compounds: Identification of a common gene signature. BMC Genomics, 16, 683. Ikeda, Y., Adachi, Y., Ishii, T., Miura, N., Tamura, H., & Ohno, N. (2008). Dissociation of Toll-like receptor 2-mediated innate immune response to Zymosan by organic solvent-treatment without loss of Dectin-1 reactivity. Biological &/and Pharmaceutical Bulletin, 31, 13–18. Jungmann, J., Rayner, J. C., & Munro, S. (1999). The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a Golgi mannosyltransferase complex. Journal of Biological Chemistry, 274, 6579–6585. Klis, F. M., Boorsma, A., & De Groot, P. W. (2006). Cell wall construction in Saccharomyces cerevisiae. Yeast, 23, 185–202. Lesage, G., Shapiro, J., Specht, C. A., Sdicu, A. M., Ménard, P., Hussein, S., et al. (2005). An interactional network of genes involved in chitin synthesis in Saccharomyces cerevisiae. BMC Genetics, 6, 8. Lesage, G., & Bussey, H. (2006). Cell wall assembly in Saccharomyces cerevisiae. Microbiology and Molecular Biology Reviews, 70, 317–343. Li, J., & Karboune, S. (2019). Characterization of the composition and the techno-functional properties of mannoproteins from Saccharomyces cerevisiae yeast cell walls. Food Chemistry, 297, 124867. https://doi.org/10.1016/j.foodchem.2019.05.141. López-Ribot, J. L., & Chaffin, W. L. (1996). Members of the Hsp70 family of proteins in the cell wall of Saccharomyces cerevisiae. Journal of Bacteriology, 178, 4724–4726. Maneesri, J., Azuma, M., Sakai, Y., Igarashi, K., Matsumoto, T., Fukuda, H., et al. (2005). Deletion of MCD4 involved in glycosylphosphatidylinositol (GPI) anchor synthesis leads to an increase in β-1,6-glucan level and a decrease in GPI-anchored protein and mannan levels in the cell wall of Saccharomyces cerevisiae. Journal of Bioscience and Bioengineering, 99, 354–360. Meirelles, A. A. D., da Cunha, R. L., & Gombert, A. K. (2018). The role of Saccharomyces cerevisiae in stabilizing emulsions of hexadecane in aqueous media. Applied Microbiology and Biotechnology, 102, 3411–3424. Novak, M., & Vetvicka, V. (2008). β-Glucans, history, and the present: Immunomodulatory aspects and mechanisms of action. Journal of Immunotoxicology, 5, 47–57. Orlean, P. (2012). Architecture and biosynthesis of the Saccharomyces cerevisiae cell wall. Genetics, 192, 775–818. Sakai, Y., Azuma, M., Takada, Y., Umeyama, T., Kaneko, A., Fujita, T., et al. (2007). Saccharomyces cerevisiae mutant displaying β-glucans on cell surface. Journal of Bioscience and Bioengineering, 103, 161–166. Takada, Y., Nishino, Y., Ito, C., Watanabe, H., Kanzaki, K., Tachibana, T., et al. (2014). Isolation and characterization of baker's yeast capable of strongly activating a macrophage. FEMS Yeast Research, 14, 261–269. Takada, Y., Mizobuchi, A., Kato, T., Kasahara, E., Ito, C., Watanabe, H., et al. (2014). Isolation of diploid baker's yeast capable of strongly activating immune cells and analyses of the cell wall structure. Bioscience, Biotechnology, and Biochemistry, 78, 911–915. Winzeler, E. A., Shoemaker, D. D., Astromoff, A., Liang, H., Anderson, K., Andre, B., et al. (1999). Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science, 285, 901–906. Young, S. H., Ye, J., Frazer, D. G., Shi, X., & Castranova, V. (2001). Molecular mechanism of tumor necrosis factor-alpha production in 1→3-β-glucan (zymosan)-activated macrophages. Journal of Biological Chemistry, 276, 20781–20787.
activity. There have been no reports that such mutations affect emulsion formation, but the accumulation of such findings is essential for the development of a yeast-derived emulsifier. The findings from this study could be useful for this development. Construction of additional promising strains is expected by, for example, combining the mutations found in this research. CRediT authorship contribution statement Shinsuke Nerome: Data curation, Formal analysis, Writing - original draft, Writing - review & editing. Masaya Onishi: Data curation, Formal analysis. Daiki Saito: Data curation, Formal analysis. Ayano Mizobuchi: Data curation, Formal analysis. Tatsuya Ando: Data curation, Formal analysis. Yui Daira: Data curation, Formal analysis. Azusa Matsumoto: Data curation, Formal analysis. Yoshihiro Ojima: Project administration, Writing - review & editing. Masayuki Azuma: Funding acquisition, Project administration, Writing - original draft, Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work was supported by funding from the Institute for Fermentation, Osaka, Japan and the Public Foundation of Elizabeth Arnold-Fuji, Japan. References Bosson, R., Jaquenoud, M., & Conzelmann, A. (2006). GUP1 of Saccharomyces cerevisiae encodes an O-acyltransferase involved in remodeling of the GPI anchor. Molecular Biology of the Cell, 17, 2636–2645. Brachmann, C. B., Davies, A., Cost, G. J., Caputo, E., Li, J. P., Hieter, & Boeke, J. D. (1998). Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast, 14, 115–132. Cabib, E., & Arroyo, J. (2013). How carbohydrates sculpt cells: Chemical control of morphogenesis in the yeast cell wall. Nature Reviews Microbiology, 11, 648–655. Cameron, D. R., Cooper, D. G., & Neufeld, R. J. (1988). The mannoprotein of Saccharomyces cerevisiae is an effective bioemulsifier. Applied and Environment Microbiology, 54, 1420–1425. Dallies, N., Francqois, J., & Paquet, V. (1998). A new method for quantitative determination of polysaccharides in the yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae. Yeast, 14, 1297–1306. De Iseppi, A., Curioni, A., Marangon, M., Vincenzi, S., Kantureeva, G., & Lomolino, G. (2019). Characterization and emulsifying properties of extracts obtained by physical and enzymatic methods from an oenological yeast strain. Journal of the Science of Food and Agriculture. https://doi.org/10.1002/jsfa.9833. De Smet, R., Allais, L., & Cuvelier, C. A. (2014). Recent advances in oral vaccine
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