Abstract: Rhizobium radiobacter WSH2601 was used as the starting strain, and a genetically stable actinomycin D-resistant mutant strain WSH-F06 was obtained by UV light and nitrosoguanidine mutagenesis. The effects of nutrients such as carbon and nitrogen sources, as well as environmental conditions such as inoculum volume, loading volume, and initial pH on the cell growth and accumulation of coenzyme Q10 of the mutant strain were investigated in shake flasks. F06 on cell growth and coenzyme Q10 accumulation. Through mutagenesis and optimization of fermentation conditions, the coenzyme Q10 production and intracellular content of the mutant strain WSH-F06 reached 34 mg/L and 2.4 mg/g, respectively, which were 16% higher than that of the starting strain under the same conditions.
1 Preface
Coenzyme Q10 (CoQ10), also known as ubiquinone, is a derivative of 2,3-dimethoxy-5-methyl-1,4-dibenzoquinone with 10 isoprenoid derivatives in the side chain. It is a natural antioxidant synthesized by cells and an activator of cellular metabolism, and has the function of enhancing immunity. It is a natural antioxidant synthesized by cells and an activator of cellular metabolism, and has the function of enhancing the immunity of the body, thus it is a biochemical drug with high clinical value.
Recent studies have shown that Coenzyme Q10 has anti-tumor effects and has been used clinically in the treatment of advanced metastatic cancer. It can also lower blood pressure and is used in the treatment of congestive heart failure. It has also been shown to be effective in the treatment of scurvy, duodenal ulcer, necrotizing periodontitis, viral hepatitis, and in promoting pancreatic function and secretion[2-4] . Coenzyme Q10 also has anti-aging properties[5] , so it has a wide range of applications in cosmetics.
Coenzyme Q10 can be synthesized by biological extraction, chemical synthesis and microbial fermentation. The production of coenzyme Q10 by microbial fermentation is the most promising method because of its abundant raw materials, mild reaction conditions, relatively simple isolation and purification process, and high clinical value of the product[1] .
At present, the main factor limiting the industrial production of coenzyme Q10 by fermentation is the low fermentation yield, which leads to the high production cost. There are few research reports on the production of coenzyme Q10 by fermentation method in China, but there is a method in Japan which involves the selection and breeding of high-yielding strains of coenzyme Q10 and their use in the production of coenzyme Q10[6] , and the coenzyme Q10 yield of the mutant strain of Soilobacillus can be up to 2.50 mg/g. The metabolic engineering research on the biosynthesis of coenzyme Q10 is under way, but due to the complexity of the synthesis pathway of coenzyme Q10 and its many steps, no breakthrough has been made[7] . However, due to the complexity of the coenzyme Q10 synthesis pathway and the many steps involved, a breakthrough has not yet been achieved[7] .
In the present work, a mutant strain of WSH-F06 was obtained by complex mutagenesis using Rhizobium radiobacter WSH2601 as the starting strain, and its fermentation culture conditions were initially investigated.
2 Materials and Methods
2.1 Strains
The starting strain was Rhizobium radiobacter WSH2601, preserved in the Environmental Biotechnology Laboratory, School of Bioengineering, Jiangnan University.
2.2 Culture media
Seed medium (g/L): glucose 20, peptone 10, yeast 10, sodium chloride 5; pH 7.2, sterilized at 121oC for 15 min. Inclined medium with 20 g/L of agar.
Bacterial basal medium (g/L): glucose 5 (NH4)2SO4 2 s sodium citrate 1 s KH2PO4 6, K2HPO4 4 s agar 20; pH 7.2 121oC sterilized for 15 min.
Basic fermentation medium (g/L): glucose 30, peptone 10, yeast paste 10, KH2PO4 0.4, Na2HPO4.12H2O 1.5, MgSO4.7H2O 0.3; pH 7.2, sterilized at 121oC for 15 min.
2.3 Strain culture methods
2.3.1 Slope activation and seed culture
Inoculate the activated slant with the inoculated strains stored in the refrigerator and incubate for 24 h at 30oC; add the activated slant into the seed medium and incubate for 24 h at 30oC with shaking at 200r/min. Shaking flasks were filled with 50 mL of 250 mL triangular flasks if not specified.
2.3.2 Fermentation cultures
The seed culture was added to the fermentation medium at 5%(j) inoculum and incubated at 30oC for 50 h with 200r/min shaking (unless otherwise specified). Three parallel samples were taken from each sample.
2.4 Coenzyme Q10 Extraction
15 mL of fermentation broth was centrifuged at 3000 r/min for 20 min, and the supernatant was poured off. Add 15 mL of acetone into the fermentation broth, and centrifuge the supernatant at 3000 r/min for 10 min with ultrasonic shaking for 0.5 h. The supernatant was analyzed by HPLC[8] .
2.5 Cell dry weight determination
4 mL of the fermentation broth was centrifuged at 10,000 r/min for 10 min and the organisms were collected and washed twice with distilled water, dried at 60oC for 48 h until constant weight and then weighed.
2.6 Mutagenesis methods
The bacteria were inoculated into seed medium, incubated in shake flasks for 24 h, and then centrifuged at 10 000 r/min for 5 min. 5 mL of the culture solution was collected, and the bacteria were washed twice with 0.1 mol/L sodium phosphate buffer (pH 6.5) to form a bacterial suspension with a concentration of 107 organisms/mL. 10 mL of the suspension was placed in a sterile dish, irradiated with ultraviolet light (UV) for 5 s, and then added with nitrosoguanidine (NTG) solution to make the concentration of NTG in the treatment solution increase. 10 mL of the suspension was placed in a sterile dish, irradiated with ultraviolet light (UV) for 5 s, and then nitrosoguanidine (NTG) solution was added to make the final concentration of NTG in the treatment solution 300 mg/L. The cells were washed three times with 0.1 mol/L phosphate buffer (pH 6.5) after NTG had been in action for 20 min. After 12 h of incubation in the seed medium, the cells were centrifuged and washed three times, diluted and spread directly on the bacterial basal medium. Then, a certain amount of actinomycin D was added in the middle of the plate and the plate was incubated at 30oC for 4~5 d. After the appearance of obvious inhibition circle, the single colony with good growth within the inhibition circle was selected as the mutant strain of actinomycin D-resistant bacteria.
3 Results and discussion
3.1 Acquisition of high yielding mutant strains
Actinomycin D (ActD) is a teratogen and carcinogen. Its structure contains a planar phenoxazine ring and two cyclic pentapeptides, which can be inserted into the DNA molecule and can selectively inhibit transcription and protein synthesis[9] . ActD is highly cytotoxic, and the addition of a certain amount of ActD to the culture medium can cause stress and toxicity to bacterial cells. Coenzyme Q10 is a physiologically active substance that enhances immunity. If an ActD-resistant mutant strain is obtained after mutagenesis, which indicates that the mutant strain is more resistant to teratogens, the intracellular accumulation of physiologically active substances, such as coenzyme Q10, may be increased.
R. radiobacter WSH2601 was used as the starting strain, and 70 actinomycin D mutant strains were obtained by combined mutagenesis with ultraviolet light and nitrosoguanidine. The mutant strains were inoculated into basic fermentation medium and screened in shake flasks. 7 mutant strains were found to increase coenzyme Q10 production by more than 10% compared with the starter strain. These 7 mutant strains were each inoculated into 3 shake flasks for re-screening, and a mutant R. radiobacter (ActDr) WSH-F06 was found to have a coenzyme Q10 yield of 11±0.2 mg/L, which was 16% higher than that of the starting strain (9.5±0.2 mg/L). The coenzyme Q10 yield of this strain was stabilized above 10.8 mg/L in each generation after five consecutive passages, which confirmed the good genetic stability of this mutant strain, and therefore it was selected as a further research strain.
3.2 Effect of carbon source on fermentation of R. radiobacter WSH-F06 auxin Q10
3.2.1 Effect of different carbon sources on coenzyme Q10 fermentation
The effect of different carbon sources on the production of coenzyme Q10 by R. radiobacter WSH-F06 fermentation was investigated by keeping the other components of the fermentation medium unchanged at a concentration of 30 g/L, and the results are shown in Fig. 1. It is shown that glucose and sucrose are favorable for the accumulation of coenzyme Q10, and the highest production of coenzyme Q10 of 11.1 mg/L was obtained when glucose was used as the carbon source.
3.2.2 Effect of glucose concentration on coenzyme Q10 fermentation
The effect of glucose concentration in the medium on the production of coenzyme Q10 is shown in Figure 2. The highest dry weight and coenzyme Q10 production were observed at a glucose concentration of 30 g/L, which reached 10.2 g/L and 10.8 mg/L, respectively. Further increase of glucose concentration inhibited the growth of the bacteria and the accumulation of coenzyme Q10.
3.2.3 Effect of Complex Carbon Sources on Coenzyme Q10 Fermentation
Although the yield of Coenzyme Q10 was not high when molasses was used as the sole carbon source, this study investigated the effect of combining glucose and molasses as carbon sources on the production of Coenzyme Q10, considering that there are many microfactors in molasses that can stimulate the growth of microorganisms. As shown in Figure 3, the addition of molasses to the glucose medium increased the production of coenzyme Q10. When 30 g/L glucose and 60 g/L molasses were added to the medium, Coenzyme Q10 production increased by 13% compared with that of 30 g/L glucose as the sole carbon source, and further increase in the amount of molasses was not favorable to the accumulation of Coenzyme Q10.
3.3 Effect of nitrogen source on fermentation of R. radiobacter WSH-F06 auxin Q10
3.3.1 Effect of different nitrogen sources on coenzyme Q10 fermentation
The effects of different nitrogen sources at a concentration of 160 mmol/L on the production of CoQ10 were investigated by using 30 g/L glucose and 60 g/L molasses as carbon sources, and the results are shown in Fig. 4. R. radiobacter (ActDr) WSH-F06 has a broad-spectrum of nitrogen utilization, with higher CoQ10 yields when using either organic nitrogen sources (peptone, yeast paste, and corn syrup) or inorganic nitrogen sources [(NH4)2SO4 and NH4NO3] as the nitrogen source s. The highest CoQ10 yield was achieved when using corn syrup as the nitrogen source, reaching 13 8 mg/L. 2SO4 and NH4NO3] were used as nitrogen sources, and the production of coenzyme Q10 was higher than that of organic nitrogen sources (peptone, yeast paste, corn syrup) and inorganic nitrogen sources [(NH4) 2SO4 and NH4NO3].
3.3.2 Nitrogen source orthogonal experiments
R. radiobacter (ActDr) WSH-F06 Higher coenzyme Q10 yields were achieved when peptone, yeast paste, corn syrup and (NH4)2SO4 were used as single nitrogen sources. In order to investigate whether a combination of nitrogen sources can further increase the yield of coenzyme Q10, an orthogonal experiment was conducted to investigate the effect of different nitrogen ratios on coenzyme Q10 yield. The orthogonal design is shown in Table 1. 30 g/L glucose and 60 g/L molasses were used as the carbon sources, and the rest of the conditions were unchanged.
In the concentration range of the experiment, peptone had the greatest effect on the accumulation of coenzyme Q10, and (NH4)2SO4 had the least effect (NH4)2SO4 7 g/L peptone 5 g/L yeast 5 g/L, corn syrup 30 g/L was the optimal nitrogen source ratio. Under this nitrogen ration, the highest yield of Auxin Q10 reached 24.1 mg/Ls with 30 g/L glucose and 60 g/L molasses as the carbon sources, and the highest yield of Auxin Q10 reached 75% higher than that of a single nitrogen source (Fig. 4) with shake flask incubation for 50 h. The highest yield of Auxin Q10 reached 24.1 mg/Ls with 30 g/L glucose and 60 g/L molasses as the carbon sources.
3.4 Effect of environmental conditions on the fermentation of R. radiobacter WSH-F06 auxin Q10
3.4.1 Effect of initial pH on coenzyme Q10 fermentation
The effect of initial pH on growth and coenzyme Q10 synthesis is shown in Fig. 5. The bacterium did not grow at the acidic initial pH, and the coenzyme Q10 production was close to zero. In the range of pH 6.8~7.5, the intracellular coenzyme Q10 content remained unchanged at about 1.9 mg/g, and there was little change in the amount of coenzyme Q10 production and the bacterial volume, so the initial pH could be chosen as 7.2.
3.4.2 Effect of loading volume on coenzyme Q10 fermentation
The different filling volumes of fermentation flasks can reflect the level of dissolved oxygen in the fermentation medium to a certain extent. The effects of different filling volumes of 500 mL flasks on the growth of the bacteria and the synthesis of coenzyme Q10 were investigated. Figure 6 shows that the highest coenzyme Q10 production was achieved at 50 mL, which indicates that R. radiobacter (ActDr) WSH-F06 has an optimal requirement of dissolved oxygen for coenzyme Q10 production, which is consistent with the findings of a previous study using the starting strain in our laboratory[10] .
3.4.3 Effect of Inoculum Volume on Coenzyme Q10 Fermentation
The effects of inoculum size on the growth and synthesis of coenzyme Q10 were investigated by adding 3%, 5%, 7%, 9% and 11% (j) to the fermentation medium at pH 7.2 (data not shown). In the range of inoculum levels, the biomass did not change much and the intracellular content and yield of coenzyme Q10 were higher at 7%~9%, so 7% inoculum level was preferred.
3.5 Shake flask fermentation of R. radiobacter WSH-F06 for coenzyme Q10 production
The optimal nutrient and environmental conditions for the fermentation of coenzyme Q10 as determined from the above shake flask experiments were 30 g/L glucose and 60 g/L molasses as the carbon source, 7 g/L (NH4)2SO4 and 5 g/L peptone, 5 g/L yeast paste, and 30 g/L maize syrup as the nitrogen source at an initial pH of 7.2, with a volume of 50 mL and an inoculum volume of 7%, and incubated in shake flasks at 30oC at 200 r/min. The culture was shaken at 200 r/min at 30oC to examine the accumulation of coenzyme Q10 by R. radiobacter WSH-F06 as shown in Figure 7.
During R. radiobacter WSH-F06 fermentation, coenzyme Q10 synthesis and cell growth were partially coupled. A small amount of coenzyme Q10 was synthesized during the logarithmic growth phase and the maximum coenzyme Q10 production and intracellular coenzyme Q10 content reached 34.0 mg/L and 2.4 mg/g, respectively, at 83 h of fermentation. When the strain was cultured in basic fermentation medium, the production reached a maximum of 11.0 mg/L at 50 h of fermentation, and then showed a decreasing trend (data not given). After optimization of the medium and environmental conditions, the fermentation time was extended to 83 h, but the coenzyme Q10 yield increased by 210% compared with the pre-optimization period, and the production intensity increased from 0.22 mg/(L.h.) to 0.41 mg/(L.h.).
The coenzyme Q10 yields of the starting strain R. radiobacter WSH2601 and the actinomycin D-resistant mutant strain R. radiobacter(ActDr)WSH-F06 were 29.3 and 34.0 mg/L, respectively, and the intracellular coenzyme Q10 contents were 2.1 and 2.4 mg/g, respectively, after incubation with the optimized medium for 83 h. This indicates that the combination of UV and NTG mutagenesis combined with the screening of actinomycin D resistance was effective. The intracellular coenzyme Q10 content was 2.1 mg/g and 2.4 mg/g, respectively. The coenzyme Q10 production of the mutant strain was increased by about 16% compared with that of the starting strain, which indicated that the combination of UV and NTG mutagenesis combined with the screening of actinomycin D resistance was effective. The effect of optimization of fermentation conditions on coenzyme Q10 yield was greater than that of mutation breeding, indicating that optimization of fermentation conditions is still an important tool to exploit the potential of strains.
Yoshida et al[6] developed a mutant strain of Agrobacterium tumefaciens AU-55 that produced 110 mg/L of coenzyme Q10 and 2.5 mg/g of intracellular coenzyme Q10, with a production intensity of 0.76 mg/(L.h.). The actinomycin-resistant D mutant strain R. radiobacter (ActDr) WSH-F06 obtained in this study showed comparable intracellular coenzyme Q10 content to that of A. tumefaciens. However, due to the high-density culture of A. tumefaciens AU-55 by replenishment during the fermentation process, its coenzyme Q10 production was higher than that of R. radiobacter (ActDr) WSH-F06.
4 CONCLUSIONS
(1) A high yielding mutant strain of coenzyme Q10, WSH-F06, was obtained by combined mutagenesis with ultraviolet light and nitrosoguanidine using Rhizobium radiobacter WSH2601 as the starting strain and actinomycin D resistance as the screening model. The nutrient and environmental conditions of the mutant strain were optimized for the production of coenzyme Q10 in shake flask experiments, and the final yield was 34 mg/L, which was 3.6 times higher than that before mutagenesis and optimization. Nutritional and environmental conditions were optimized for the production of coenzyme Q10 by the mutant strain in shake flask experiments, and the final coenzyme Q10 yield was 34 mg/L, which was 3.6 times higher than that of the mutant strain before mutagenesis and optimization. The intracellular coenzyme Q10 content of the mutant strain WSH-F06 was 2.4 mg/g, which was comparable to that of A. tumefaciens AU-55, a mutant strain of A. tumefaciens developed by Yoshida[6] .
(2) Actinomycin D resistance was used as a screening model for selecting mutant strains with high coenzyme Q10 production, which provides a new idea for mutation breeding of coenzyme Q10 and other physiologically active substances-producing bacteria.
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