Mutagenesis selection and fermentation optimization of extracellular glutathione-producing brewer's yeasts

 Glutathione (GSH), or γ-glutamyl-L-cysteine-glycine, is an amino acid composed of three amino acids (glutamate, cysteine, and glycine) that are synthesized in the enzyme glutamylcysteine synthetase and glutathione synthase.

GSH is a non-protein thiol tripeptide synthesized under continuous action [1], which has an important role in protecting cells against antioxidant damage and maintaining intracellular homeostasis [2-4].GSH is widely used in industrial fields such as food [5-7], cosmetic [8], and pharmaceuticals [9-12], and it can be used as antioxidant and flavoring agent to prevent browning of food and to maintain its unique flavor [13-16], as well as free radical scavenger, with the effects of protecting kidney and enhancing liver detoxification [17-18]. It can also be used as a free radical scavenger, which can protect the kidney and enhance the detoxification ability of the liver [17-18].

 

Microbial fermentation is currently the most common method for industrial production of GSH [19-20], however, as an intracellular product, the productivity and economic benefits of GSH extraction downstream of fermentation cannot effectively meet the market demand, which restricts the industrialization of GSH to a certain extent, if the intracellular GSH of the fermenting strains can be efficiently secreted to the extracellular, the purified GSH can be prepared directly in the fermentation broth, simplifying the extraction process and reducing the difficulty of downstream isolation and purification of GSH, thus improving the productivity of GSH. If intracellular GSH can be effectively secreted to extracellular, then GSH can be directly prepared and purified in the fermentation broth, which simplifies the extraction process, reduces the difficulty of downstream isolation and purification of GSH, and thus improves GSH productivity. In recent years, how to obtain extracellular GSH-producing fermentation strains has attracted the attention of scholars at home and abroad[21] . For example, the use of genetic engineering technology to enhance the extracellular transport of GSH by fermentation strains[22-24] , or to improve the permeability of the cell membrane to promote the exocytosis and accumulation of glutathione. However, the modification cost and technical requirements for the construction of extracellularly fermented glutathione engineering bacteria are high, and the regulation of cell membrane permeability is an organic reagent chemical permeation method [25], which is not suitable for industrial production considering the impact on the growth activity of the strains and the environment.

 

Using traditional UV mutagenesis, not only can the target strains be selected easily and rapidly, but also can ensure the genetic stability of the mutant strains [26], however, in the current study, only intracellular glutathione-producing mutant strains were screened by UV mutagenesis, and no extracellular glutathione-producing mutant strains were obtained [27-29]. Therefore, in this study, we used Saccharmyces cere- visiae GAQ4 as the starting strain, and selected a genetically stable mutant strain with high extracellular glutathione production by UV mutagenesis, and further improved the extracellular GSH production by optimizing the fermentation conditions, so as to provide a new reference for the strategy of extracellular fermentation of GSH and the realization of the industrialized extracellular fermentation and production of GSH. We also optimized the fermentation conditions to further increase the extracellular GSH production.

 

1 Materials and Methods

1.1 Materials and reagents

Saccharomyces cerevisiae GAQ4: industrial fermentation in the Key Laboratory of the Ministry of Education, Hubei University of Technology, Hubei Provincial Collaborative Innovation Center, Laboratory A610.

Glucose, yeast powder, peptone, ammonium sulfate, potassium dihydrogen phosphate, anhydrous magnesium sulfate, agar powder, phosphoric acid (all analytically pure): Sinopharm Chemical Reagent Corporation; Sodium 1-heptanesulfonate, Glutathione (all analytically pure): Shanghai McLean Biochemistry Technology Co.

 

1.2 Instruments and equipment

SW-CJ-1D Single Purification Workbench: Suzhou Purification Equipment Co., Ltd; ZSD-12 Automatic Biochemical Incubator: Shanghai Zhicheng Analytical Instrument Co. Sartorius Universal PH Meter (PB-10): Sartorius (Shanghai) Trading Co., Ltd; Electrothermal Constant Temperature Blast Drying Oven: Shanghai Jinghong Experimental Equipment Co.

 

1.3 Test methods

1.3.1 Cultivation methods

Seed liquid medium: glucose 20 g/L, peptone 20 g/L, baking powder 10 g/L, pH natural.

Seed solid medium: glucose 20 g/L, peptone 20 g/L, baking powder 10 g/L, agar 20 g/L, pH natural.

Fermentation medium: glucose 20 g/L, yeast 10 g/L, ammonium sulfate 8 g/L, potassium dihydrogen phosphate 3 g/L, magnesium sulfate 0.15 g/L, pH natural.

Seed culture: Take the bacterial suspension preserved in glycerol tubes and insert it into the seed medium, and incubate it at 30 ℃ and 180 r/min of shaking speed.

Fermentation culture: Take the seed sap in logarithmic growth period and put it into the fermentation medium with an inoculum volume of 10% by volume, and incubate it under the temperature of 30 ℃ and the rotational speed of 180 r/min of the shaker.

 

1.3.2 Ultraviolet mutagenesis

Mutagenesis process: Preheat 15 W UV lamp for 20 min. Take the seed bacterial suspension in logarithmic growth period, dilute the bacterial suspension to the cell number of 107 CFU/mL~108 CFU/mL, take 200 μL of the dilution solution and spread it onto the seed solid medium, then immediately place the medium plate at a distance of 30 cm from the UV lamp for UV irradiation, the irradiation time was 10, 20, 30, 40, 50, and 60 s, and each group was set up in 3 parallels, and the number of single bacterial colonies grown on each plate was counted. Immediately after irradiation, the plates were placed upside down in an incubator at 30 ℃ to avoid light, and the number of single colonies grown on each plate was counted. Diluted bacterial suspension without UV irradiation was spread on solid medium and then incubated under light as control group. Calculate the UV mutagenic lethality, take the UV irradiation time as the horizontal coordinate and the UV mutagenic lethality as the vertical coordinate, and plot the UV mutagenic lethality curve. The formula for calculating the mutagenic lethality is as follows.

 

Mutagenic lethality/% = (number of colonies on control plate - number of colonies on mutagenized plate)/number of colonies on control plate × 100.

Iterative UV mutagenesis: Using the extracellular GSH content of the strain as the screening index, GAQ4 was subjected to iterative UV mutagenesis, i.e., the mutant strains that met the screening indexes after the previous mutation in the next mutation were taken as the starting strains and then subjected to mutation screening again. Three parallel groups were set for each mutation to obtain sufficient number of mutants and increase the positive mutation rate. Each UV mutagenesis should be plotted with the UV mutagenic lethal curve in order to select the optimal mutagenic dose for each generation of mutant strains.

 

Genetic stability test: In order to confirm that the obtained target strains have good genetic stability, the target strains screened were successively passed for 8 times, and the fermentation culture was carried out on the 1st, 4th and 8th generations, and the extracellular glutathione content of the fermentation production was measured against the control of the starting strain GAQ4. 1.3.3 One-way test of fermentation conditions

A one-way test was conducted to investigate the effects of fermentation period, shaking flask volume, initial pH, fermentation temperature and shaking flask speed on the extracellular GSH content produced by shaking flask fermentation of the mutant strains, using extracellular GSH content as an index, and the experimental levels were: fermentation period of 24, 36, 48, 60, 72 h; shaking flask volume of 50, 75, 100, 125, 150 mL/300 mL; initial pH 5.0, 5.5, 6.0, 6.5, 7.0; fermentation temperature 26, 30, 34, 38, 42 ℃; fermentation speed 120, 140, 160, 180, 200 r. The optimization of the fermentation conditions of the mutant strain was carried out in three parallels, and the average values of each factor were taken to determine the optimal fermentation conditions of the shaking flasks.

 

1.3.4 Measurement of biomass

A certain volume of bacterial suspension was centrifuged at 8000 r/min for 5 min to collect the wet bacterial body, and the bacterial body was washed with sterile water for 3 times, and then dried in an oven at 65 ℃ until constant weight, and then weighed to obtain the dry weight of the cells, and the biomass was calculated by the following formula.

Biomass/(g/L) = cell dry weight (g) / volume of bacterial suspension (L)

 

1.3.5 Determination of glutathione

The GSH content was determined by high performance liquid chr- omatography (HPLC) [30]. The chromatographic conditions were as follows: Inertsil ODS-SP C18 column, sodium heptanesulfonate-phosphate buffer solution (1-heptanesulfonate 6.8 g, potassium dihydrogen phosphate 2.2 g, pH 3.0 adjusted with phosphoric acid, methanol (phosphate buffer 90:10), flow rate 1.0 mL/min, injection volume 90:10. The mobile phases were sodium heptanesulfonate-phosphate buffer solution (6.8 g of sodium 1-heptanesulfonate, 2.2 g of potassium dihydrogen phosphate, pH 3.0 adjusted with phosphoric acid, and the volume of ultrapure water was adjusted to 1 L) and methanol (the volume ratio of phosphate buffer to methanol was 90:10), with the flow rate of 1.0 mL/min, the injection volume of 20 μL, and the ultraviolet (UV) detection wavelength was 210 nm.

Weigh 10 mg of glutathione standard, diluted to 10 mL with ultrapure water, and then diluted to the concentrations of 200, 400, 600, 800 and 1 000 mg/L. The peak areas corresponding to the different GSH concentrations were detected by HPLC, and the standard curve was plotted by HPLC with the GSH concentration as the horizontal coordinate and the peak area as the vertical coordinate, and the regression equation of the standard curve was y=18 045x+168 919, R2 =0.999 3. The standard curve was plotted with GSH concentration as the horizontal coordinate and peak area as the vertical coordinate (Figure 1), and the regression equation of the standard curve was y=18 045x+168 919, R2 =0.999 3, which was used for the calculation of GSH content.

 

2 Results and analysis

2.1 Mutagenesis breeding results

 

2.1.1 Extracellular GSH Content of Departure Strain GAQ4

The extracellular glutathione content of GAQ4 was 7.37 mg/L after shaking flask fermentation for 48 h. The extracellular glutathione content of GAQ4 was used as an indicator of the starting strain, and the extracellular glutathione content of GAQ4 was compared with that of the mutant obtained by mutagenesis.

 

2.1.2 Results of UV mutagenesis screening

Using the extracellular GSH content as the screening index, the starting strain GAQ4 was subjected to UV iterative mutagenesis according to the method of 1.3.2. The results of the first UV mutagenesis screening are shown in Fig. 2.

A total of 200 single colonies were picked out after the first UV irradiation treatment, and 15 mutant strains were selected from the primary screening strains based on the high extracellular glutathione content after fermentation (Fig. 2B), among which the extracellular glutathione content of the mutant UV1-6 was 13.87 mg/L, which increased by 88.20% compared with that of the starting strain, and was selected to continue the screening by UV mutagenesis. The mutant strain UV1-6 was selected to continue the UV mutagenesis screening.

 

The mutant UV1-6 was used as the starting strain for the second UV mutagenesis, and the results of the second UV mutagenesis screening are shown in Fig. 3. From Fig. 3A, it can be seen that the mutant strain showed a lethal rate of 78.6% when irradiated with UV for 30 s, which is higher than that of the previous mutagenesis at the same dose, so we still chose to use the same dose of UV irradiation for 30 s for the second mutation treatment.

As shown in Fig. 3B, 14 mutant strains with relatively high extracellular glutathione content were selected after the second UV mutagenesis screening, among which the extracellular glutathione content of mutant UV2-2 was 16.10 mg/L, which was 118.45% and 16.08% higher than that of the starting strain GAQ4 and mutant UV1-6, respectively.

The mutant UV2-2 was subjected to a third UV mutagenesis treatment, and the results are shown in Fig. 4.

 

As can be seen from Fig. 4A, the lethality of the third UV mutagenesis was obviously increased after the same mutagenic dose, and the lethality reached 69.5% at 10 s of UV irradiation, and 81.9% at 20 s of UV irradiation, so 15 s of UV irradiation was selected as the time for the third mutation treatment. After screening, 11 mutants with high extracellular GSH content were obtained. As shown in Figure 4B, the extracellular GSH content of the mutant UV3-10 was 19.08 mg/L, which was 158.89%, 37.56% and 18.51% higher than that of the starting strain, the mutant UV1-6 and the mutant UV2-2, respectively. Through the above analysis, the positive mutation effect of the third mutagenesis was not as obvious as that of the previous two, and the extracellular glutathione content of only a small number of mutants screened was increased, and the content of some mutants was even lower than that before the third mutagenesis, and the lethality of UV mutagenesis under the same mutagenic dose gradually increased, which was beyond the range of lethality of high-probability positive mutation, therefore, we did not carry out the screening of the fourth mutagenesis, and we did not select three UV mutations. Therefore, the mutant strains UV1-6, UV2-2 and UV3-10 obtained from the third UV mutagenesis screening were selected for the next test.

 

2.1.3 Genetic stability results

The mutant strains UV 1-6, UV 2-2 and UV 3-10 obtained from three UV mutagenesis screening were successively passed for eight times, and the extracellular glutathione contents were determined after the fermentation culture of the 1st, 4th and 8th generations, respectively.

The highest extracellular glutathione content of 18.56 mg/L was found in UV 3-10 at the 8th generation, and the fermentation performance of UV 3-10 in the production of extracellular glutathione was genetically stable, so UV 3-10 was selected as the target strain.

 

2.2 Optimization of fermentation conditions

Under the original shaking flask fermentation conditions, i.e., shaking flask filling volume of 100 mL/300 mL, natural initial pH, fermentation temperature of 30 ℃, and rotational speed of 180 r/min, the extracellular GSH content of the mutant strain was 18.56 mg/L, and the biomass was 5.64 g/L. One-factor optimization experiments were carried out to optimize the fermentation period, shaking flask filling volume, initial pH, fermentation temperature, rotational speed, and the fermentation time of the mutant strain respectively. The fermentation cycle, shake flask loading, initial pH value, fermentation temperature and shake flask rotation speed were optimized by one-factor optimization test. The effects of fermentation period on the extracellular glutathione production by the target strain UV3-10 are shown in Figure 5.

 

As shown in Figure 5, the extracellular glutathione content of the mutant strain increased and then decreased with the extension of fermentation time. The extracellular glutathione content of the mutant strain was 18.97 mg/L at the fermentation time of 48 h. The extracellular glutathione content began to decrease with the extension of the fermentation time, so the fermentation time of 48 h was chosen as the fermentation time, and the other fermentation conditions were further optimized on the basis of the fermentation time.

The effect of shake bottle loading on extracellular glutathione production by the target strain is shown in Figure 6.

 

 From Fig. 6, it can be seen that the highest extracellular glutathione content of the target strain was found when the liquid volume of shaking flask was 75 mL/300 mL, and as the liquid volume of shaking flask gradually increased, the extracellular glutathione content began to decline, and the high liquid volume of shaking flask affected the dissolved oxygen in the shaking flask, thus affecting the fermentation of the target strain, so the optimal shaking flask liquid volume of 75 mL/300 mL was chosen.

The results of the optimization of the initial pH value of the shaker bottle of the mutant strain are shown in Fig. 7.

 

When the initial pH value was increased from 5.0 to 6.0, the extracellular glutathione content did not change much, but increased to 6.5, the extracellular glutathione content increased rapidly to 36.42 mg/L, and then started to decrease with the increase of pH value, and reached the maximum value at pH 6.5. Therefore, the optimal initial pH value of 6.5 was chosen.

The effect of fermentation temperature on extracellular glutathione production by the target strain is shown in Figure 8.

 

 As shown in Figure 8, the effect of fermentation temperature on the extracellular glutathione content of the target strain was higher than that of other factors, and the increase in fermentation temperature promoted the secretion of extracellular glutathione by the mutant strain[31] . The extracellular glutathione content of the mutant strain increased with increasing fermentation temperature, and reached 40.63 mg/L at 34 ℃, which was 1.68 times higher than that at 30 ℃. When the fermentation temperature was increased to 34 ℃, the extracellular glutathione content reached 40.63 mg/L, which was 1.68 times of the fermentation temperature of 30 ℃. When the fermentation temperature was increased to 42 ℃, the extracellular glutathione content of the mutant strain reached 17.15 mg/L, and the biomass of the mutant strain dropped to the lowest level, which was 3.16 g/L. Therefore, the optimal fermentation temperature was selected to be 34 ℃.

The effect of shake flask speed on extracellular glutathione production by the target strain is shown in Figure 9.

 

The amount of dissolved oxygen in shake flasks was affected by the amount of liquid loaded into the flasks and the rotational speed, and the shear force generated by the rotational speed of the flasks, the mass transfer rate and the amount of liquid loaded into the flasks had an effect on the inoculum amount, the growth status and the synthesis and accumulation of the fermentation products of the strains, which can be seen in Fig. 9, the highest extracellular glutathione content was found in the flasks at the rotational speed of 140 r/min, and the target strains yielded the highest extracellular glutathione content of 52.05 mg/L, 2.80 times of the content of the optimized strain. The extracellular glutathione content of the target strain was 52.05 mg/L, which was 2.80 times higher than that before optimization.

 

After the optimization of shake flask fermentation conditions, the mutant strain reached 52.05 mg/L of extracellular GSH after 48 h of fermentation, which was 2.80 times higher than that before the optimization, with a shaking flask filling volume of 75 mL/300 mL, an initial pH value of 6.5, a fermentation temperature of 34 ℃, and a rotational speed of 140 r/min.

 

3. Discussion and conclusions

In this study, the mutant strain UV3-10 was screened for the production of extracellular GSH by iterative UV mutagenesis, and its extracellular GSH content was 18.56 mg/L. The optimal conditions for shaking flask fermentation were obtained by a one-way test, which included a fermentation period of 48 h, a loading volume of 75 mL/300 mL, an initial pH value of 6.5, a fermentation temperature of 34 ℃, and a rotational speed of 140 r/min. After the optimization, the extracellular GSH content of the mutant strain was 52.05 mg/L, which was 2.80 times higher than that before optimization.

 

The extracellular secretion mechanism of the mutant strain UV3-10 was proposed as follows: the cell permeability of the mutant strain was altered after mutagenesis, i.e., the activity of some GSH exporting proteins was strengthened or the activity of some GSH degrading proteins was inhibited after mutagenesis, so the intracellular substances of the yeast cells overflowed to the extracellular specificity, and the extracellular accumulation of GSH was generated under normal fermentation conditions. The relationship between the activity of glutathione transport proteins and GSH synthetase system of the mutant strain and its extracellular secretion mechanism can be investigated in the future, and how to maintain the stability of extracellular GSH in the downstream of fermentation is also an issue to be investigated.

 

The mutant strain UV3-10 can secret intracellularly synthesized GSH to extracellularly, thus realizing the effective accumulation of extracellular GSH, which greatly improves the possibility of industrial preparation of GSH in fermentation broth, and reduces the difficulty of purification and isolation compared with intracellular GSH extraction, thus saving the production cost and energy consumption. In addition, Saccharomyces cerevisiae is a food-safety strain, which is highly favored by food developers, and the results of the study on the production of extracellular GSH by fermentation of Saccharomyces cerevisiae provide new prospects and economic benefits for its application in food processing, storage and preservation, and functional condiments.

 

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