Ergosterol supplementation improves furfural tolerance of Saccharomyces cerevisiae to produce ethanol and its underlying mechanism

Abstract

Furfural produced during lignocellulose pretreatment to reduce the recalcitrance inhibits the growth of Saccharomyces cerevisiae and reduces ethanol yield. To reduce the adverse effect of furfural on S. cerevisiae, exogenous ergosterol was supplemented and the impact on S. cerevisiae under furfural stress was studied. The lag phage was shortened by 50%, and the maximum ethanol yield was increased by 158% with 50 mg/L ergosterol supplementation under 4 g/L furfural stress. Flow cytometry analysis results showed that permeable cells and intracellular reactive oxygen species were decreased by 45 and 53%, respectively with the addition of ergosterol under furfural stress. The fatty acid composition of S. cerevisiae was changed; the intracellular glycerol and ergosterol content was increased after ergosterol supplementation. The saturation of fatty acid was increased. Addition of ergosterol promoted cell growth by decreasing oxidative stress. Under 4 g/L furfural stress, the lag phage of S. cerevisiae BY4741 (erg3△) and S. cerevisiae BY4741 (erg5△) was longer than that of S. cerevisiae BY4741, and the maximum ethanol concentration was decreased.

Full Article

Ergosterol Supplementation Improves Furfural Tolerance of Saccharomyces cerevisiae to Produce Ethanol and Its Underlying Mechanism

Youpiao Jia,^a Qingyan Zhang,^a Jun Dai,^a Xueyun Zheng,^a Xianzhi Meng,^d Ranran Zhou,^a Haitao Yang,^c Lan Yao,^a,b,* and Xiong Chen ^a,*

Furfural produced during lignocellulose pretreatment to reduce the recalcitrance inhibits the growth of Saccharomyces cerevisiae and reduces ethanol yield. To reduce the adverse effect of furfural on S. cerevisiae, exogenous ergosterol was supplemented and the impact on S. cerevisiae under furfural stress was studied. The lag phage was shortened by 50%, and the maximum ethanol yield was increased by 158% with 50 mg/L ergosterol supplementation under 4 g/L furfural stress. Flow cytometry analysis results showed that permeable cells and intracellular reactive oxygen species were decreased by 45 and 53%, respectively with the addition of ergosterol under furfural stress. The fatty acid composition of S. cerevisiae was changed; the intracellular glycerol and ergosterol content was increased after ergosterol supplementation. The saturation of fatty acid was increased. Addition of ergosterol promoted cell growth by decreasing oxidative stress. Under 4 g/L furfural stress, the lag phage of S. cerevisiae BY4741 (erg3△) and S. cerevisiae BY4741 (erg5△) was longer than that of S. cerevisiae BY4741, and the maximum ethanol concentration was decreased.

DOI: 10.15376/biores.18.1.228-246

Keywords: Saccharomyces cerevisiae; Furfural; Ergosterol; Ethanol; Flow cytometry

Contact information: a: Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education & Hubei Province), College of Bioengineering, Hubei University of Technology, 28th of Nanli Road, Wuhan 430068, China; b: Collaborative Grant-in-Aid of the HBUT National “111” Center for Cellular Regulation and Molecular Pharmaceutics, Hubei University of Technology, Wuhan 430068, China; c: College of Material and Chemistry Engineering, Hubei University of Technology, 28^th of Nanli Road, Wuhan 430068, China; d: Department of Chemical and Biomolecular Engineering, University of Tennessee Knoxville, Knoxville, TN 37996-2200, USA;

*Corresponding author: yaolislan1982@aliyun.com; cx163_qx@163.com

INTRODUCTION

The process of biofuel production from lignocellulose includes pretreatment, enzymatic hydrolysis, fermentation, and distillation (Yao et al. 2022). During biomass pretreatment, various inhibitors such as furfural are generated, and these suppress the growth of Saccharomyces cerevisiae and consequently limit the ethanol production (Huang et al. 2019). Furfural is one of the main toxic chemicals formed from carbohydrates via pentose dehydration during acid-catalyzed biomass pretreatments. It prevents the growth of yeast and interferes with the critical fermentation step, thus affecting the production of bioethanol (Liu et al. 2021).

Earlier studies showed that furfural of 3 g/L reduced the production of ethanol by blocking cell growth (Gonçalves et al. 2015). S. cerevisiae oxidizes furfural to furoic acid with oxygen or reduces it to furfural alcohol without oxygen to achieve the purpose of detoxification (Wang et al. 2016). Therefore, it has been widely used in ethanol fermentation. Under stress conditions, such as ethanol at high concentration and lignocellulose hydrolysate, reactive oxygen species (ROS) accumulate in cells, which hinders their growth and fermentation (Jayakody and Jin 2021). Previously, the addition of 3.5% biochar (as an additive) was shown to improve the ethanol production of Zymomonas mobilis with furfural at high concentration (Wang et al. 2020). The addition of proline and inositol to the medium has a protective effect on the growth of S. cerevisiae under furfural stress, and the intracellular ROS is decreased with the increase of proline content (Wang et al. 2015). Furthermore, the yeast’s tolerance to acetic acid is improved by adding zinc sulfate to the culture medium (Wan et al. 2015).

Under furfural stress, cell integrity plays a vital role in maintaining cell viability and normal metabolic function. Furfural stress conditions affect the lipid content of the cell membrane (Lopes et al. 2015). Supplementation with oleic acid and ergosterol relieves the oxidative stress of S. cerevisiae (Landolfo et al. 2010).

As a structural analogue to cholesterol, ergosterol plays vital role in fungal plasma membrane (Beni et al. 2014). It regulates the permeability of the plasma membrane and the activity of proteins for membrane-binding (Wollam and Antebi 2011). It is widely applied in medicine, and its derivatives have notable antitumor activities (Kitchawalit et al. 2014). Although ergosterol biosynthesis process in yeast has been clarified, the role of ergosterol in yeast under furfural stress is unclear.

The effects of ergosterol on furfural tolerance of S. cerevisiae were assessed in the present study. To examine the underlying mechanism, the response of the cell to furfural stress was observed. The effects of ergosterol on furfural tolerance of S. cerevisiae were demonstrated by the determination and comparison of the content of glycerol, hydrogen peroxide, and catalase activity. Finally, the key genes erg3△ and erg5△ knockout strains of ergosterol synthesis were used to validate the effect of ergosterol on furfural tolerance of S. cerevisiae.

EXPERIMENTAL

Microorganism

Saccharomyces cerevisiae (isolated and given by Angel Yeast Co., Ltd), S. cerevisiae BY4741 (American type culture collection 18824), S. cerevisiae BY4741 (erg3△) (engineered and stored in our own lab), and S. cerevisiae BY4741 (erg5△) (engineered and stored in our own lab) were maintained in slants (glucose 20 g/L, agar 20 g/L, peptone 10 g/L, yeast extract 5 g/L) at 4 °C.

Different concentrations of furfural (0.0, 1.0, 2.0, 4.0 g/L) were added to the yeast extract peptone dextrose (YEPD) medium to evaluate the potential impact of furfural on S. cerevisiae. Ergosterol supplementation under furfural stress (4.0 g/L) was evaluated with different concentrations (0 mg/L, 5 mg/L, 10 mg/L, 20 mg/L, 50 mg/L).

Precultures were grown overnight at 30 ℃ in 250 mL shake flasks containing 50 mL YEPD medium without furfural and ergosterol. The initial OD600 of 0.5 was cultivated in an orbital shaker at 30 ℃ and 200 rpm with different addition of furfural with or without ergosterol supplementation.

Methods

Determination of biomass, glucose, and ethanol

The concentration of yeast biomass was determined by the measurement of absorbance at 600 nm. Glucose content was measured by the DNS method (Dai et al. 2020). Ethanol was determined with biological sensor analyzer (SBA-40C, Biological Institute of Shandong Academy of Science, Shandong province, China).

Determination of furfural

Furfural was analyzed by high performance liquid chromatography (Shimadzu LC-20A, Kyoto, Japan) with a UV detector at a wavelength of 277 nm. Furfural was separated on a C18 column (SinoChrom ODS-AP 300A 5µm), 0.1% (v/v) acetic acid was applied as eluent, at a flow rate of 1.0 mL/min.

Flow cytometry analysis

A flow cytometer (FACS, Becton-Dickinson, Franklin Lakes, NJ, USA) was used to monitor the physiological stress response of S. cerevisiae under furfural stress and ergosterol supplementation. Propidium iodide (PI, P-1304) was applied to monitor the membrane integrity, and 2′,7′-dichlorofluorescein diacetate (DCFDA) was used to determine the accumulation of ROS.

One milliliter of fermentation broth with OD₆₀₀ 0.25 was centrifuged at 2000 rpm for 5 min to collect the cells. The cells were washed three times with PBS. Pre-cooled 70% (v/v) ethanol was added to fix the cell at 4 ℃ for 1 h, and then 1 mL of PBS was added to resuspend the cells. After filtering with a 400-mesh screen, the cells were mixed with 10 μL of corresponding dye solution. The cells were protected from light at 4 ℃ for 30 min and ultrasoniced for 1 min before flow cytometry analysis.

Extraction and determination of ergosterol

The precipitate was separated from 10 mL of fermentation broth by centrifugation followed by washing with distilled water (5 mL✕2). Approximately 20 mL of 20% (v/v) sodium hydroxide solution and 10 mL of 95% (v/v) ethanol were added. After reflux saponification in a water bath for 1.5 h at 85 to 90 ℃, another 4 mL of 95% (v/v) ethanol was added, and saponification was continued for 1 h. After adding 25 mL of acetone, the mixture was shaken violently for 15 min and then left to incubate without shaking for 2 h. The upper extract was detected by a UV spectrophotometer at 282 nm (Pastinen et al. 2017).

Determination of fatty acids

The methods used for harvesting, saponification, methylation, and extraction of cellular fatty acids followed published protocols (Dai et al. 2014). The fatty acid composition analysis of the extracted yeast cells was carried out using an Agilent gas chromatograph (6890 N, Palo Alto, CA, USA) and the MIDI Sherlock YEAST6 program. The temperature of the hydrogen ion flame detector (FID) was 300 ℃. A Hp-ultra2 capillary column was used, and the temperature of the vaporization chamber was 250 ℃. The carrier gas was 30 mL/min (H₂). The furnace temperature rose from 170 °C to 260 °C at a rate of 5 °C/min.

Determination of glycerol, hydrogen peroxide, and catalase

The concentration of H₂O₂ was quantified using a hydrogen peroxide detection kit (BC3590, Beijing Solarbio Technology Co., Ltd, Beijing, China). Catalase was determined by a catalase detection kit (A007-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The concentration of glycerol was detected by a glycerol detection kit (Beijing pulley Gene Technology Co., Ltd., EL002, Beijing, China).

Statistical analysis

All experiments were performed in triplicate, and the mean ± standard deviation values were determined. The cells were collected at mid-exponential phase, unless stated otherwise. The diagrams were drawn by OriginPro 2019b software (Northampton, MA, USA) and FlowJo_V10. Statistica 23.0 software (SPSS Inc., Chicago, IL, USA) was applied for significant difference analysis.

RESULTS AND DISCUSSION

Effect of Ergosterol Supplementation on Furfural Tolerance of S. cerevisiae

Influence of furfural on growth and ethanol production of S. cerevisiae

To investigate the impact of furfural on the growth of S. cerevisiae, different concentrations of furfural were added to the YEPD medium. The addition of 1 g/L furfural did not show obvious influence on the growth of S. cerevisiae. When the furfural concentration was increased to 2 and 4 g/L, the lag phase was extended from 4 h (1 g/L furfural) to 12 (2 g/L furfural) and 48 h (4 g/L furfural), respectively, as shown in Fig. 1A. The X_max was decreased under higher furfural stress, as illustrated in Table 1. Notably, S. cerevisiae began to grow when the furfural concentration was decreased to 0.1 g/L regardless of the initial concentration. Figure 1C shows that the concentration of furfural decreased sharply during the first 10 h. The rate of decrease slowed until all furfurals were converted.

The glucose consumption and ethanol production profile are presented in Fig. 1B and 1D, respectively. When S. cerevisiae began to grow, glucose concentration decreased sharply, and ethanol production increased. Furfural in a low concentration (1 g/L) did not exhibit significant impact on glucose consumption and ethanol production of S. cerevisiae. At higher concentrations, furfural showed a great impact on the ethanol production of S. cerevisiae. The EtOH_max was 9 g/L (control), 8.75 g/L (1 g/L), 7.00 g/L (2 g/L), and 5.50 g/L (4 g/L), respectively. The time corresponding to EtOH_max was delayed from 8 h (control) to 12 h (1 g/L), 24 h (2 g/L), and 48 h (4 g/L). The higher concentration of furfural had a larger impact on ethanol production from S. cerevisiae.

Previous studies have shown that when furfural is less than 2.5 g/L, it has no inhibitory effect on ethanol fermentation of highly active dry yeast. The increasing furfural concentration slows furfural metabolism, reduces ethanol yield, and prolongs the fermentation cycle. A previous study showed that ethanol yield is reduced to 87% at 32 h after addition of 5 g/L of furfural (Fu et al. 2014).

Table 1. Kinetic Parameters for S. cerevisiae in YEPD Medium with Increasing Initial Furfural Concentrations

Fig. 1. Cultures profiles of S. cerevisiae in YEPD medium with increasing initial furfural concentrations. (A, OD₆₀₀; B, ethanol; C, furfural concentration; D, glucose)

Ergosterol supplementation increased furfural tolerance of S. cerevisiae

At 4 g/L furfural loading, different concentrations of initial ergosterol were added into the medium to analyze the influence of ergosterol supplementation on furfural tolerance of S. cerevisiae. As shown in Fig. 2A and Table 2, the lag phase was shortened with the increased ergosterol supplementation, which was 48 h (0 mg/L), 36 h (5 mg/L, 10 mg/L), and 24 h (20 mg/L, 50 mg/L), respectively. In addition, the X_max was also increased with increased ergosterol supplementation. S. cerevisiae began to grow when the furfural concentration was decreased to 0.1 g/L. The results implied that ergosterol supplementation might help speed up furfural detoxification (Fig. 2C).

The glucose consumption and ethanol production profile with ergosterol supplementation at 4 g/L furfural loading are presented in Figs. 2B and 2D. With the growth of S. cerevisiae, glucose decreased, and ethanol was increased accordingly. Ergosterol supplementation shortened the lag phase and increased the ethanol yield. The EtOH_max was 6.0, 6.2, 6.9, 9.7, and 15.5 g/L, for control, 5 mg/L, 10 mg/L, 20 mg/L, and 50 mg/L ergosterol supplementation, respectively. The results indicated that ergosterol supplementation increased ethanol production of S. cerevisiae under furfural stress (4 g/L).

Fig. 2. Cultures profiles of S. cerevisiae in YEPD media containing 4 g/L furfural and different ergosterol supplementation. (A, OD₆₀₀; B, ethanol; C, furfural concentration; D, glucose)

Table 2. Kinetic Parameters for S. cerevisiae in YEPD Media with Increasing Initial Ergosterol Concentrations under 4g/L Furfural Stress

Supplementing components in the medium (lauric acid, palmitoleic acid, stearic acid, linolenic acid, and ergosterol) can change the lipid composition of cells, and different lipid composition is closely related to the performance of S. cerevisiae at low temperatures (Marian et al. 2009). After supplementing oil, the fermentation cycle is reduced, which are consistent with the present results. Landolfo et al. (2010) found that supplementing lipid nutrients under adverse conditions reduces the oxidative stress and oxidative damage to S. cerevisiae.

To determine whether the addition of ergosterol promotes the production of ethanol without furfural, only ergosterol was added to the YEPD medium. As shown in Fig. 3A, compared with the control, adding ergosterol of different concentrations increased the biomass (OD₆₀₀) of S. cerevisiae. The maximum biomass increased with the increase of ergosterol concentration. Figure 3B shows that when ergosterol was added, compared with control, the yield of ethanol was not increased. When 50 mg/L ergosterol was added, glucose was exhausted within 6 h, two hours earlier than the other groups (Fig. 3C). The results confirmed that the addition of ergosterol improved the furfural tolerance of yeast. More importantly, the ethanol yield of S. cerevisiae was improved under high furfural stress.

Fig. 3. S. cerevisiae batch cultures profiles in YEPD media containing increasing initial ergosterol concentrations. (A, OD₆₀₀; B, ethanol; C, glucose)

Analysis of cell membrane porosity of S. cerevisiae under furfural stress with ergosterol supplementation

To elucidate the mechanism of ergosterol enhancing furfural tolerance of S. cerevisiae, cell membrane porosity was determined and compared with furfural at 4 g/L with 50 mg/L ergosterol. As shown in Fig. 4, propidium iodide (PI) was applied to evaluate the porosity of the membrane. The membrane permeability of control was 0.13%, which was increased to 15% with 4 g/L furfural, indicating that cells with permeable cell membrane were increased under furfural stress. The proportion of cells with incomplete cell membrane decreased to 8.5% with ergosterol supplementation, which was 45% lower than that of the furfural group.

The results indicated that the membrane integrity of yeast cells was affected by the addition of furfural. Ergosterol supplementation improved cell membrane integrity under furfural stress. In previous studies, under 4 g/L furfural stress, the proportion of permeabilized cells of S. carlsbergensis increased to 57%, and the proportion of healthy cells in the highest concentration furfural group was the lowest at the end of culture. The integrity of the cell membrane was influenced by the presence of furfural (Lopes et al. 2015). Additionally, furfural had a serious impact on the membrane potential. The membrane potential of S. cerevisiae was low in the lag phase. The decrease of membrane potential was the greatest when furfural, vanillin, and acetic acid were present (López et al. 2021). In this study, similar results were obtained.

Fig. 4. S. cerevisiae subpopulations under 4 g/L furfural stress and 50 mg/L ergosterol. (PI)， “***” (P＜0.001)

Analysis of ROS of S. cerevisiae under furfural stress with ergosterol supplementation

ROS accumulation leads to DNA and protein damage, and reduced viability of cultured cells (Moradas et al. 1996). As shown in Fig. 5, the proportion of cells with ROS under furfural stress was 28%, and this was decreased to 13% with ergosterol addition, indicating that the ROS level was decreased with ergosterol supplementation. The proportion of cells with ROS decreased by 53% after ergosterol addition. Earlier studies indicated that lipid nutrients could reduce cell-to-cell variations under oxidative stress, and intracellular ROS content was lower (Landolfo et al 2010).

ROS accumulated gradually in the lag phase and growth phase with acetic acid, furfural, vanillin, and other inhibitors. Compared with furfural and vanillin, greater ROS accumulation was observed with acetic acid (López et al. 2021). Furfural-induced ROS accumulation in S. cerevisiae was studied by other researchers (Allen et al 2010). Compared with 50 mM furfural, the damage induced by furfural was not very serious with 25 mM furfural.

Fig. 5. Effect of ergosterol supplementation on intracellular ROS content in S. cerevisiae under furfural stress, “**” (0.001≤P＜0.01)

Regulation of total fatty acid composition of S. cerevisiae under furfural stress with ergosterol supplementation

The results showed that supplementation with lipid nutrients (ergosterol) reduced the oxidative stress of S. cerevisiae. Fatty acid composition under furfural stress should be studied to explore the mechanism of ergosterol supplementation.

The fatty acids in S. cerevisiae mainly include palmitoleic acid (C16:1), palmitic acid (C16:0), oleic acid (C18:1), myristic acid (C14:0), lauric acid (C12:0) and myristic acid (C14:1), as shown in Table 3. The content of main unsaturated fatty acids in S. cerevisiae cells was 88%. Under 4 g/L furfural stress, the content of main unsaturated fatty acids in S. cerevisiae increased to 93%. After supplementation with 50 mg/L ergosterol, the content of main unsaturated fatty acids was 85%.

Table 3. Effect of Ergosterol Supplementation on Total Fatty Acid Composition of S. cerevisiae under Furfural Stress

Compared with the control, exogenous addition of furfural and ergosterol changed the composition of fatty acids of S. cerevisiae. When furfural was added, the ratio of unsaturated fatty acids increased, resulting in an increase of 71% of unsaturated/saturated fatty acids and a decrease in cell membrane fluidity. When ergosterol was added, the proportion of unsaturated fatty acids decreased by 26%. The proportion of saturation of fatty acids increased, resulting in increased stability of cell membrane.

The increase of lipid nutrients (ergosterol) can maintain the stability of the membrane. The ergosterol content of yeast cells was determined and compared, as shown in Fig. 6A. In the control group, the ergosterol content of cells in logarithmic phase and stationary phase was 0.27% and 0.43% respectively, which was comparable under furfural stress. The ergosterol contents in cells of logarithmic phase and stationary phase after supplementation with ergosterol were increased to 0.49% and 0.91% respectively. This result indicated that exogenous addition of ergosterol increased the ergosterol content, thus changing the fatty acid composition. It has been reported that the proportion of ergosterol in cell membrane of S. cerevisiae increases under vanillin stress (Ayako et al. 2009). The increase of ergosterol content in cell membrane could improve the thermal stress resistance of S. cerevisiae (Caspeta et al. 2014). Ergosterol and unsaturated lipids may cooperate with each other to maintain the optimal membrane thickness, which can protect membrane proteins from ethanol (Vanegas et al. 2014).

Fig. 6. Effect of ergosterol supplementation on A, ergosterol content; B, glycerol content of S. cerevisiae under furfural stress, “**” (0.001≤P＜0.01), “***” (P＜0.001). To be specific, the exponential phase was evaluated at the beginning of this phase.

Glycerol is another important component in the cell membrane of S. cerevisiae under stress. As shown in Fig. 6B, normally, the accumulation of glycerol in the logarithmic phase (12 h) was 18.9 mg/L. The cells grow rapidly in the logarithmic phase, and the intracellular glycerol begins to be consumed as a carbon source to support the growth of cells, such as the synthesis of lipids or other protective substances (Ansell et al. 1997). With 4 g/L furfural, the intracellular glycerol content of S. cerevisiae was lower in logarithmic phase. Excessive NADH is re-oxidized during furfural detoxification, resulting in reduced glycerol content (Liu et al. 2020). With 50 mg/L ergosterol supplementation, the intracellular glycerol content in logarithmic growth stage was nearly 5 times higher than that under furfural stress. Metabolomics research shows that yeast respond to furfural stress by regulating glycerol synthesis, improving glycolytic activity (Jung et al. 2017).

An earlier study found that S. cerevisiae 1200 was more tolerant to ethanol than S. cerevisiae AJL2155 (Chi and Arneborg 1999). The proportion of unsaturated fatty acids of S. cerevisiae 1200 and the ratio of ergosterol to phospholipid was much higher than that of S. cerevisiae AJL 2155. The fermentation ability of S. cerevisiae was improved after cultured with supplementation of different lipids (Marian et al. 2009). The fermentation time of cells added with palmitoleic acid (C16:1) was reduced, and the viability of cells was higher. S. cerevisiae was cultured with different fatty acids and ergosterol to change the lipid composition of the membrane. The supplementation of ergosterol did not show impact on the total content of fatty acids, but it increased unsaturated fatty acids. Another study showed that in order to resist salt stress, the ergosterol content of Zygosaccharomyces rouxii was increased to maintain the stability of cell membrane, reduce the fluidity of cell membrane and increase the proportion of unsaturated fatty acids, resulting in the increase of the proportion of unsaturated / saturated fatty acids (Wang et al. 2020), which was found in the present study.

Changes of CAT and hydrogen peroxide under furfural stress with ergosterol supplementation

The antioxidant defense system of S. cerevisiae is composed of super oxide dismutase and catalase (CAT), which catalyze superoxide anion in cytoplasm and mitochondria to produce hydrogen peroxide and oxygen, and then decompose hydrogen peroxide into water and oxygen (Li et al. 2020). When cells are damaged by ROS, S. cerevisiae forms a defense by inducing catalase (Eleutherio et al. 2017). As shown in Fig. 7, CAT with 4 g/L furfural was higher than that with 4 g/L furfural and 50 mg/L ergosterol.

Fig. 7. Effect of ergosterol supplementation on CAT and hydrogen peroxide content in S. cerevisiae under furfural stress, “***” (P＜0.001). To be specific, the exponential phase was evaluated at the beginning of this phase.

The content of extracellular hydrogen peroxide was much higher without ergosterol under furfural stress in lag phase, indicating that the addition of ergosterol alleviated the oxidative stress of yeast cells. That might be one of the reasons that ergosterol supplementation could shorten the lag phase of S. cerevisiae under furfural stress. The ability of TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-l-oxyl) to scavenge intracellular ROS in S. cerevisiae was studied by Li et al. (2020). The addition of TEMPOL enhances cell growth and increased the CAT activity, resulting in higher H₂O₂ tolerance. The overexpression of YCR102C promotes the growth and ethanol production of S. cerevisiae under acetic acid stress. Through the expression of YCR102C, the activity of CAT was increased by 47% under 5 g/L acetic acid stress (Chen et al. 2019).

erg△mutants exhibit varying furfural sensitivity

Ergosterol plays an important role in stress adaption. Ergosterol levels are closely related to the ability of yeast to tolerate in low temperature, low sugar, high alcohol, and other stress (Hu et al. 2017; Kamthan et al. 2017). Ergosterol biosynthesis is complex, involving many enzymes and energy. The last steps of ergosterol biosynthesis are catalyzed by ERG3/ERG4/ERG5. Although mutations of these genes do not affect yeast growth, they do have impact on the composition of the cell membrane, thus influencing stress tolerance (Kodedová et al. 2015).

Fig. 8. Batch cultures profiles of different S. cerevisiae in YEPD media, curve with square refers to S. cerevisiae BY4741, curve with round represents S. cerevisiae BY4741 erg3△, curve with triangle is S. cerevisiae BY4741 erg5△ (A, OD₆₀₀; B, ethanol; C, furfural concentration; D, glucose)

Fig. 9. Batch cultures profiles of different S. cerevisiae in YEPD medium under 4g/L furfural concentrations, curve with square refers to S. cerevisiae BY4741, curve with round represents S. cerevisiae BY4741 erg3△, curve with triangle is S. cerevisiae BY4741 erg5△ (A, OD₆₀₀; B, ethanol; C, furfural concentration; D, glucose)

To further verify the effect of ergosterol in S. cerevisiae under furfural stress, ergosterol synthase knockout strains (S. cerevisiae BY4741 (erg3△) and S. cerevisiae BY4741 (erg5△)) were applied. The results in Fig. 8, Fig. 9, and Table 4 showed that without furfural stress, the kinetic parameters of the three strains were comparable. The value of μ was 0.042 h^-1, X_max was around 41.00 g/L, and Y_P/S was about 0.56 g/g.

The lag phase of S. cerevisiae BY4741 was 36 h under 4 g/L furfural stress. The best ethanol performance was 6.1 g/L at 48 h. Furfural was converted, and glucose was consumed completely within 48 h. S. cerevisiae BY4741 (erg3△) began to grow after 52 h. The best ethanol performance was 5.6 g/L at 60 h.

Table 4. Kinetic Parameters for Different S. cerevisiae in YEPD Media

Furfural was converted and glucose was consumed completely within 60 h. Compared with S. cerevisiae BY4741, the time required for furfural degradation was significantly longer. The corresponding time of the maximum ethanol concentration of S. cerevisiae BY4741 (erg3△) was 12 h later than that of S. cerevisiae BY4741. The maximum ethanol concentration was 8.6% lower than that of the original strain.

With 4 g/L furfural, S. cerevisiae BY4741 (erg5△) began to grow after 52 h. The best ethanol performance was 5.30 g/L at 60 h. Furfural was completely converted within 58 h, which was significantly longer than that of the original strain. The corresponding time of the maximum ethanol concentration was 12 h longer than that of the original strain, and the maximum ethanol concentration was 14% lower than that of the original strain.

The deletion of genes (ERG6, ERG 2, ERG 3, ERG 5, and ERG 4) in the final step of ergosterol biosynthesis had an impact on the physiological function of S. cerevisiae plasma membrane. Most ERG mutants are more sensitive to various stress than wild-type ones. The sensitivity was positively correlated with the hyperpolarization of plasma membrane. Under NaCl stress, erg2△, erg3△ and erg6△ mutants are the most sensitive to acid stress (Kodedová et al. 2015). Erg3△ knockout strain BS-1, a key gene for ergosterol synthesis, was constructed. Compared with the original strain, BS-1 showed lower tolerance to salt stress. It was found that mutants of S. cerevisiae (erg2△, erg3△, erg4△, erg5△, erg6△, erg24△, erg28△) were sensitive to acid (Kawahata et al. 2006).

CONCLUSIONS

1. The addition of ergosterol can shorten the lag period and increase the content of ethanol in the presence of high concentration furfural (4 g/L) of S. cerevisiae.
2. Ergosterol supplementation could decrease membrane permeability. When ergosterol was added under furfural stress, the proportion of unsaturated fatty acids was decreased, the proportion of unsaturated/saturated fatty acids was decreased, which increased the stability of cell membrane and ensured the fluidity of cell membrane.
3. 3. Under 4 g/L furfural stress, the lag period of S. cerevisiae BY4741 (erg3△) and S. cerevisiae BY4741 (erg5△) was longer than that of the original strain, and the ethanol content decreased. It can be confirmed that ergosterol has a great effect on the furfural tolerance of S. cerevisiae.

ACKNOWLEDGMENTS

The authors are grateful for the support from the National Natural Science Foundation of China (No. 21978074 and 31871789), the China Scholarship Council (No. 2011842330 and 201508420257), key project of Hubei Provincial Department of Education (D20211404), and key Laboratory of Fermentation Engineering (Ministry of Education) (No.202105FE04).

REFERENCES CITED

Allen, A., Clark, W., McCaffery, J., Cai, Z., Lanctot, A., Slininger, P., Liu, Z., and Gorsich, S. (2010). “Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae,” Biotechnol. Biofuels 3, 2-0. DOI: 10.1186/1754-6834-3-2

Ansell, R., Granath, K., Hohmann, S., Thevelein, J., and Adler, L. (1997). “The two isoenzymes for yeast NAD+-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaptation and redox regulation,” EMBO J. 16, 2179-2187. DOI: 10.1093/emboj/16.9.2179

Ayako, E., Toshihide, N., and Jun, S. (2009). “Involvement of ergosterol in tolerance to vanillin, a potential inhibitor of bioethanol fermentation in Saccharomyces cerevisiae,” Fems. Microbiol. Lett. 299, 95-99. DOI: 10.1111/j.1574-6968.2009.01733.x

Beni, A., Soki, E., Lajtha, K., and Fekete, I. (2014). “An optimized HPLC method for soil fungal biomass determination and its application to a detritus manipulation study,” J. Microbiol. Meth. 103, 124-130. DOI: 10.1016/j.mimet.2014.05.022

Caspeta, L., Chen, Y., Ghiaci, P., Feizi, A., Buskov, S., Hallstrom, B., Petranovic, D., and Nielsen, J. (2014). “Altered sterol composition renders yeast thermotolerant,” Science 346(6205), 75-78. DOI: 10.1126/science.1258137

Chen, H., Li, J., Wan, C., Fang, Q., Bai, F., and Zhao, X. (2019). “Improvement of inhibitor tolerance in Saccharomyces cerevisiae by overexpression of the quinone oxidoreductase family gene YCR102C,” FEMS. Yeast Res. foz055. DOI: 10.1093/femsyr/foz055

Chi, Z., and Arneborg, N. (1999). “Relationship between lipid composition frequency of ethanol-induced respiratory deficient mutants and ethanol tolerance in Saccharomyces cerevisiae,” J. Microbiol. 86, 1047-1052. DOI: 10.1046/j.1365-2672.1999.00793.x

Dai, J., Li, K., Song, N., Yao, W., Xia, H., Yang, Q., Zhang, X., Li, X., Wang, Z., Yao, L., Yang, S., and Chen, X. (2020). “Zygosaccharomyces rouxii, an Aromatic yeast isolated from chili sauce, is able to biosynthesize 2-phenylethanol via the Shikimate or Ehrlich pathways,” Frontiers in Microbiology 11, article no. 597454. DOI: 10.3389/fmicb.2020.597454

Dai, J., Xu, M., Peng, F., Jiang, F., Chen, X., Wang, Z., and Fang, C. (2014). “Pontibacter soli sp. nov., isolated from the soil of a Populus rhizosphere in Xinjiang, China,” Antonie Van Leeuwenhoek 105, 65-72. DOI: 10.1007/s10482-013-0053-8

Eleutherio, E., Brasil, A., França, M., Almeida, D., Rona, G., and Magalhães, R. (2017). “Oxidative stress and aging: Learning from yeast lessons,” Fungal Biol-UK S187861461730168X. DOI: 10.1016/j.funbio.2017.12.003

Fu, S., Hu, J., and Liu, H. (2014). “Inhibitory effects of biomass degradation products on ethanol fermentation and a strategy to overcome them,” BioResources 9, 4323-4335. DOI: 10.15376/biores.9.3.4323-4335

Gonçalves, F., Santos, E., and Macedo, G. (2015). “Alcoholic fermentation of Saccharomyces cerevisiae, Pichia stipitis and Zymomonas mobilis in the presence of inhibitory compounds and seawater,” J. Basic Microb. 55, 695-708. DOI: 10.1002/jobm.201400589

Huang, S., Liu, T., Peng, B., and Geng A. (2019). “Enhanced ethanol production from industrial lignocellulose hydrolysates by a hydrolysate-cofermenting Saccharomyces cerevisiae strain,” Bioprocess Biosyst. Eng. 42, 883-896. DOI: 10.1007/s00449-019-02090-0

Hu, Z., He, B., Ma, L., Sun, Y., Niu, Y., and Zeng, B. (2017). “Recent advances in ergosterol biosynthesis and regulation mechanisms in Saccharomyces cerevisiae,” Indian J. Microbiol. 57, 270-277. DOI: 10.1007/s12088-017-0657-1

Jayakody, L., and Jin, Y. (2021). “In-depth understanding of molecular mechanisms of aldehyde toxicity to engineer robust Saccharomyces cerevisiae,” Appl. Microbiol. Biot. 105, 2675-2692. DOI: 10.1007/s00253-021-11213-1

Jung, Y., Kim, S., Yang, J., Seo, J., and Kim, K. (2017). “Intracellular metabolite profiling of Saccharomyces cerevisiae evolved under furfural,” Microbial Biotechnology 10, 395-404. DOI: 10.1111/1751-7915.12465

Kamthan, A., Kamthan, M., and Datta, A. (2017). “Expression of C-5 steroldesaturase from an edible mushroom in fisson yeast enhances its ethanol and thermotolerance,” PLOS One 12, e0173381. DOI: 10.1371/journal.pone.0173381

Kawahata, M., Masak,i K., Fujii, T., and Iefuji, H. (2006). “Yeast genes involved in response to lactic acid and acetic acid: Acidic conditions caused by the organic acids in Saccharomyces cerevisiae cultures induce expression of intracellular metal metabolism genes regulated by Aft1p,” FEMS. Yeast Res. 6, article no. 924936. DOI: 10.1111/j.1567-1364.2006.00089.x

Kitchawalit, S., Kanokmedhakul, K., Kanokmedhakul, S., and Soytong, K. (2014). “A new benzyl ester and ergosterol derivatives from the fungus Gymnoascus reessii,” Nat. Prod. Res. 28, 1045-1051. DOI: 10.1080/14786419.2014.903478

Kodedová, M., Sychrová, H., and Soveral, G. (2015). “Changes in the sterol composition of the plasma membrane affect membrane potential, salt tolerance and the activity of multidrug resistance pumps in Saccharomyces cerevisiae,” PLOS One 10, e0139306. DOI: 10.1371/journal.pone.0139306

Landolfo, S., Zara, G., Zara, S., Budroni, M., Ciani, M., and Mannazzu, I. (2010). “Oleic acid and ergosterol supplementation mitigates oxidative stress in wine strains of Saccharomyces cerevisiae,” Int. J. Food Microbiol. 141, 229-235. DOI: 10.1016/j.ijfoodmicro.2010.05.020

Li, K., Zhang, J., Liu, C., Aamer, M., and Bai, F. (2020). “Elucidating the molecular mechanism of TEMPOL-mediated improvement on tolerance under oxidative stress in Saccharomyces cerevisiae,” Chem. Eng. Sci. 211, article no. 115306. DOI: 10.1016/j.ces.2019.115306

Liu, C., Li, K., Li, K., Sakdaronnarong, C., Mehmood, A., Zhao, X., and Bai, F. (2020). “Intracellular redox perturbation in Saccharomyces cerevisiae improved furfural tolerance and enhanced cellulosic bioethanol production,” Front. Bioeng. Biotechnol. 8, 615. DOI: 10.3389/fbioe.2020.00615

Liu, Z. (2021). “Reasons for 2-furaldehyde and 5-hydroxymethyl-2-furaldehyde resistance in Saccharomyces cerevisiae: Current state of knowledge and perspectives for further improvements,” Appl. Microbiol. Biot. 105, 2991-3007. DOI: 10.1007/s00253-021-11256-4.

Lopes, T., Santo, R., Reis, A., and Passarinho, P. (2015). “Effect of furfural on Saccharomyces carlsbergensis growth, physiology and ethanol production,” Appl. Biochem. Biotechnol. 182, 708-720. DOI: 10.1007/s12010-016-2356-5

López, P., Peng, C., Arneborg, N., Junicke, H., and Gernaey, K. (2021). “Analysis of the response of the cell membrane of Saccharomyces cerevisiae during the detoxification of common lignocellulosic inhibitors,” Sci. Rep. 11. DOI: 10.1038/s41598-021-86135-z

Marian, R., José, M., Albert, M., and Nicolas, R. (2009). “Effect of lipid supplementation upon Saccharomyces cerevisiae lipid composition and fermentation performance at low temperature,” Biotechnol. Biofuels 228, 833-840. DOI: 10.1007/s00217-008-0996-6

Moradas, F., Costa, V., Piper, P., and Mager, W. (1996). “The molecular defenses against reactive oxygen species in yeast,” Mol. Microbiol. 19, 651-658. DOI: 10.1046/j.1365-2958.1996.403940.x

Pastinen, O., Nyyssölä, A., Pihlajaniemi, V., and Sipponen, M. (2017). “Fractionation process for the protective isolation of ergosterol and trehalose from microbial biomass,” Process Biochem. 217-223. DOI: 10.1016/j.procbio.2017.04.002

Vanegas, J., Contreras, M., Faller, R., and Longo, M. (2014). “Role of unsaturated lipid and ergosterol in ethanol tolerance of model yeast biomembranes,” Biophys. J. 102, 507-516. DOI: 10.1016/j.bpj.2011.12.038

Wan, C., Zhang, M., Fang, Q., Xiong, L., Zhao, X., Hasunuma, T., Bai, F., and Kondo, A. (2015). “The impact of zinc sulfate addition on the dynamic metabolic profiling of Saccharomyces cerevisiae subjected to long term acetic acid stress treatment and identification of key metabolites involved in the antioxidant effect of zinc,” Metallomics 7, 322-332. DOI: 10.1039/C4MT00275J

Wang, D., Zhang, M., Huang, J., Zhou, R., Jin, Y., and Wu, C. (2020). “Zygosaccharomyces rouxii combats salt stress by maintaining cell membrane structure and functionality,” J. Microbiol. Biotechn. 1, 62-70. DOI: 10.4014/jmb.1904.04006

Wang, S., He, Z., and Yuan, Q. (2016). “Xylose enhances furfural tolerance in Candida tropicalis by improving NADH recycle,” Chem. Eng. Sci. 158, 37-40. DOI: 10.1016/j.ces.2016.09.026

Wang, W., Dai, L., Wu, B., Qi, B., Huang, T., Hu, G., and He, M. (2020). “Biochar-mediated enhanced ethanol fermentation (BMEEF) in Zymomonas mobilis under furfural and acetic acid stress,” Biotechnol. Biofuels 13, 28-39. DOI: 10.1186/s13068-020-1666-6

Wang, X., Bai, X., Chen, D., Chen, F., Li, B., and Yuan, Y. (2015). “Increasing proline and myo-inositol improves tolerance of Saccharomyces cerevisiae to the mixture of multiple lignocellulose-derived inhibitors,” Biotechnol. Biofuels 8, 142-154. DOI: 10.1186/s13068-015-0329-5

Wollam, J., and Antebi, A. (2011). “Sterol regulation of metabolism, homeostasis, and development,” Annu. Rev. Biochem. 80, 885-916. DOI: 10.1146/annurev-biochem-081308-165917

Yao, L., Cui, P., Chen, X., Yoo, C., Liu, Q., Meng, X., Xiong, L., Ragauskas, A., and Yang, H. (2022). “A combination of deep eutectic solvent and ethanol pretreatment for synergistic delignification and enhanced enzymatic hydrolysis for biorefinery process,” Bioresource Technol. 350, 126885-126892. DOI: 10.1016/j.biortech.2022.126885

Article submitted: August 1, 2022; Peer review completed: October 15, 2022; Revised version received: October 25, 2022; Accepted: October 26, 2022; Published: November 8, 2022.

DOI: 10.15376/biores.18.1.228-246