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Abstract In the present study， single factors including fermentation temperature， inoculate amount， fermentation duration， and ratio of fermentation medium volume to total flask volume （dissolved oxygen tension） were optimized for enhancing the production of coenzyme Q10from genetic engineered Rhodobacter sphaeroides overexpressing UbiG. The experimental results suggested that optimal single factors were： inoculate amount 2%， fermentation temperature 30 ℃， fermentation duration 48 h， and ratio of fermentation medium volume to total flask volume 80%. The present study will promote the large scale production of CoQ10from microorganisms.
Key words CoQ10; Rhodobacter sphaeroides; Genetic engineering; Optimization; Fermentation
Coenzyme Q10（CoQ10） is a lipidsoluble material and widespread in both prokaryotes and eukaryotes， which exhibits multiple functions such as resisting oxidation and transferring electron in the electron transport chain[1]. Moreover， a lot of researches have been suggested that CoQ10is effective to treatment many diseases. Jahangard and coworkers[2]suggested that CoQ10might be considered a safe and effective strategy for treatment of patients with bipolar disorder during their depressive phase. Chen and coworkers[3]described that CoQ10could serve as an AMPK activator and regulate the hepatic lipid metabolism to inhibit the abnormal accumulation of hepatic lipids and prevent nonalcoholic fatty liver disease progression. Moreover， CoQ10benefits the treatment for patients with fibromyalgia[4]， early chronic Peyronies disease[5]， metabolic syndrome[6]. Currently， CoQ10is widely used in food， comestic and pharmaceutical industry and the requirement of CoQ10is increasing. Microbial fermentation is one of most promising way to produce natural functional CoQ10. Among which， Rhodobacter sphaeroides is proven to be an excellent microorganism for producing natural functional CoQ10[7]. In the present study， we optimized the production procedure for CoQ10production from the genetic engineered R. sphaeroides overexpressing UbiG， which is an oxygenmethyltransferase， participating in two steps for the synthesis of CoQ10in R. sphaeroides. UbiG first catalyzes 2decaprenyl6hydroxyphenol into 2decaprenyl6methoxyphenol， and catalyzes the 2decaprenyl3methyl5hydroxy6methoxy1，4benzoquinone into CoQ10， which is the last step for the formation of CoQ10in R. Sphaeroides[8]. Materials and Methods
Bacterial strain growth conditions
R. sphaeroides strains were grown at 30 ℃ in malate minimal medium in dark[9]. Growth under microaerobic conditions in the dark was performed as described in our previous study[10]. 1.5μg/ml tetracycline was used for cultivation of genetic engineered R. sphaeroides overexpressing UbiG.
Effects of inoculate amount on crude CoQ10production from genetic engineered R. sphaeroides strain
A single colony was inoculated into a 50ml flask containing 40 ml of malate minimal medium and grown under microaerobic growth conditions in dark at 30 ℃ until OD660reached 0.6. Then， precultures were inoculated into 100ml flasks containing 80 ml of malate minimal medium at the ratio of 1%， 2%， 3%， 4% and 5%， respectively. The cultures were grown under microaerobic conditions in dark at 30 ℃ for 48 h. Crude CoQ10was then respectively extracted from the cell cultures and quantified as described in our previous study[11]. The experiment was repeated three times.
Effects of incubation temperature on crude CoQ10production from genetic engineered R. sphaeroides strain
A single colony was inoculated into a 50ml flask containing 40 ml of malate minimal medium and grown under microaerobic conditions in dark at 30 ℃ until OD660reached 0.6. Then， the precultures were inoculated into 100ml flasks containing 80 ml of malate minimal medium at the ratio of 1∶ 50 and grown at microaerobic conditions in dark at 27， 30 and 33 ℃ for 48 h. CoQ10was respectively extracted from the cell cultures and quantified as described in our previous study[11]. The experiment was repeated three times.
Effects of fermentation duration on crude CoQ10production from genetic engineered R. sphaeroides strain
A single colony was inoculated into a 50ml flask containing 40 ml of malate minimal medium and grown under microaerobic conditions in dark at 30 ℃ until OD660reached 0.6. Then， precultures were respectively inoculated into 100ml flasks containing 80 ml of malate minimal medium at the ratio of 1∶ 50 and grown microaerobic conditions in dark at 30 ℃ for 36， 48， 60， 72 and 84 h.CoQ10was respectively extracted from the cell cultures and quantified as described in our previous study[11]. The experiment was repeated three times.
Effects of oxygen tension （fermentation medium volume to total flask volume） on crude CoQ10production from genetic engineered R. sphaeroides strain A single colony was inoculated into a 50ml flask containing 40 ml of malate minimal medium and grown under microaerobic conditions in dark at 30 ℃ until OD660reached 0.6. Then， precultures were respectively inoculated into five 100ml flasks containing 50， 60， 70， 80 and 90 ml of malate minimal medium at the ratio of 1∶ 50and grown under microaerobic conditions in dark at 30 ℃ for 48 h.Oxygen tension in the different shaken flasks with different volumes of medium was different caused by the steady rotation speed. CoQ10was extracted from the cell cultures and quantified as mentioned above， respectively. The experiment was repeated three times.
Results and Discussion
Optimization of fermentation time
The optimized fermentation time for the enhancement of crude CoQ10from genetic engineered strain R. sphaeroides overexpressing UbiG was tested， as observed in Fig. 1. The production of CoQ10was increased with the increase of fermentation duration from 24 to 48 h. The production of crude CoQ10extracted from the strain fermented for 24， 36 and 48 h， was 21.96， 26.99 and 40.49 mg/L， respectively. Compared with the production of crude CoQ10from the cell cultures fermented for 24 and 36 h， the production of crude CoQ10from the cell cultures incubated for 48 h was increased by 84.40% and 33.36%， respectively. However， the production of crude CoQ10was declined when fermented for more than 48 h. The production of crude CoQ10from the cell cultures incubated for 60 and 72 h was 21.17 and 13.64 mg/L， respectively， which were decreased by 47.71% and 66.33% compared with the CoQ10production extracted from the cell cultures incubated for 48 h， respectively. It is reasonable to speculate that fermentation duration for 48 h was the optimized fermentation duration， which was similar to optimized incubation during for the genetic engineered R. sphaeroides overexpressing UbiE as described in our previous study[10].
Optimization of inoculate amount
The experimental results for optimizing the inoculate amount for enhancement of crude CoQ10from genetic engineered R. sphaeroides overexpressing UbiG is revealed in Fig. 2. Obviously， the inoculate amount greatly affected the crude CoQ10production from genetic engineering R. sphaeroides overexpressing UbiG. The productionof crude CoQ10was 18.14， 39.17， 29.31， 22.75 and 24.56 mg/L when the inoculate amount was 1%， 2%， 4%， 6% and 8%， respectively. The production of crude CoQ10was highest when the inoculate amount was 2%， which was over 2 times of the crude CoQ10production when the inoculate amount was 1%. The crude CoQ10production was decreased by 25.17%， 41.93% and 37.29% when the inoculate amount was 4%， 6% and 8%， respectively compared with the crude CoQ10production at the inoculate amount of 2%. Optimization of fermentation temperature
The fermentation temperature was optimized for enhancement of crude CoQ10production from the genetic engineered strain R. sphaeroides overexpressing UbiG， as seen in Fig. 3. It could be seen that from 23 to 30 ℃ with the increase of the incubation temperature， the production of crude CoQ10from the genetic engineered R. sphaeroides overexpressing UbiG was increased. However， with the increase of incubation temperature from 30 to 37 ℃， the production of crude CoQ10from the genetic engineered R. sphaeroides overexpressing UbiG was decreased. It was similar with the genetic engineered R. sphaeroides overexpressing UbiE[10]. Clearly， 30 ℃ was the optimized temperature for production of crude CoQ10from the genetic engineered strain， with the yield of 39.17 mg/L. Compared with the crude CoQ10production at 23 and 27 ℃， the CoQ10production at 30 ℃ was increased by 509.36% and 27.93%， respectively. While， the production of CoQ10production at 33 and 37 ℃ was decreased by 39.88% and 50.66%， respectively.
Agricultural Biotechnology 2020
Optimization of fermentation medium volume used in shaken flasks （changes dissolved oxygen tension）
The dissolved oxygen tension will be affected by filling different volumes of medium in the same volume flasks when shaken at steady speed. The ubiG gene is initiated by the puf operon promoter which is oxygen tension dependent[12]. When the dissolved oxygen tension is high， the PpsR repressor will be bound to the puf promoter and overexpression of ubiG will be repressed. However， at low dissolved oxygen tension， the PpsR repressor will be bound with the antirepressor protein AppA and form a complex， and thus the PpsR repressor cannot bind the puf promoter[13]. Consequently， the overexpression of ubiG will be activated. Effect of oxygen tension on the production of crude CoQ10is shown in Fig. 4. The highest production of CoQ10was obtained when the medium volume to whole flask volume was 80%， suggesting the optimized oxygen tension was obtained for growth and fermentation. The production of crude CoQ10was 11.02， 18.85， 26.62， 37.21 and 31.63mg/L， respectively when the filled medium volume was 50%， 60%， 70%， 80% and 90% of the total flask volume. When the volumes were lower than 80%， the dissolved oxygen tension in the medium was relatively higher and thus the puf promoter activity was repressed. On the other hand， the bacteria need oxygen for survival， so when the medium volume was 90%， the production of crude CoQ10was lower than that of 80%. It was concluded that dissolved oxygen tension was crucial for the production of crude CoQ10from the genetic engineering strains. Conclusion
The optimal processes for producing CoQ10from genetic engineered R. sphaeroides overexpressing UbiG were： fermentation duration 48 h， inoculate amount 2%， fermentation temperature 30 ℃， and volume of fermentation medium to flask total volume 80%.
References
[1] RIZVI A， CHIBBER S， NASEEM I. Cu（II）vitamin D interaction leads to free radicalmediumted cellular DNA damage： a novel putative mechanism for its selective cytotoxic action against malignant cells[J]. Tumour Biol， 2015， 36（3）： 1695-1700.
[2] JAHANGARD L， YASREBIFAR F， HAGHIGHI M， et al. Influence of adjuvant Coenzyme Q10on inflammatory and oxidative stress biomarkers in patients with bipolar disorders during the depressive episode[J]. Molecular Biology Reports， 2019， 46（5）： 5333-5343.
[3] CHEN K， CHEN X， XUE H， et al. Coenzyme Q10attenuates highfat dietinduced nonalcoholic fatty liver disease through activation of the AMPK pathway[J]. Food Funct， 2019， 10（2）： 814-823.
[4] CORDERO MD， MORENOFERNANDEZ AM， DEMIGUEL M， et al. Coenzyme Q10distribution in blood is altered in patients with fibromyalgia[J]. Clin Biochem， 2009， 42（7-8）： 732-735.
[5] SAFARINEJAD MR. Safety and efficacy of coenzyme Q10supplementation in early chronic Peyronies disease： a doubleblind， placebocontrolled randomized study[J]. Int J Impot Res， 2010， 22（5）： 298-309.
[6] RAYGAN F， REZAVANDI Z， DADKHAH TEHRANI S， et al. The effects of coenzyme Q10administration on glucose homeostasis parameters， lipid profiles， biomarkers of inflammation and oxidative stress in patients with metabolic syndrome[J]. Eur J Nutr， 2016， 55（8）： 2357-2364.
[7] LU W， SHI Y， HE S， et al. Enhanced production of CoQ10by constitutive overexpression of 3demethyl ubiquinone9 3methyltransferase under tac promoter in Rhodobacter sphaeroides[J]. Biochemical Engineering Journal， 2017（72）： 42-47.
[8] LU W， YE L， LV X， et al. Identification and elimination of metabolic bottlenecks in the quinone modification pathway for enhanced coenzyme Q10production in Rhodobacter sphaeroides[J]. Metab Eng， 2015（29）： 208-216.
[9] REMES B， BERGHOFF BA， FORSTNER KU， et al. Role of oxygen and the OxyR protein in the response to iron limitation in Rhodobacter sphaeroides[J]. BMC Genomics， 2014（15）： 794.
[10] TANG K， ZHAO ZZ， ZHANG L， et al. Optimization of coenzyme Q10production procedure from Rhodobacter sphaeroides overexpressing UbiE[J]. Agricultural Biotechnology， 2019， 8（5）： 19-20， 25.
[11] TANG K， WANG J， WANG W， et al. Production of functional coenzyme Q10from genetic engineered Rhodobacter sphaeroides[J]. Advance Journal of Food Science and Technology， 2019， 17（3）： 48-53.
[12] BOWMAN WC， DU S， BAUER CE， et al. In vitro activation and repression of photosynthesis gene transcription in Rhodobacter capsulatus[J]. Mol Microbiol， 1999， 33（2）： 429-437.
[13] MASUDA S， BAUER CE. AppA is a blue light photoreceptor that antirepresses photosynthesis gene expression in Rhodobacter sphaeroides[J]. Cell， 2002， 110（5）： 613-623.
Key words CoQ10; Rhodobacter sphaeroides; Genetic engineering; Optimization; Fermentation
Coenzyme Q10（CoQ10） is a lipidsoluble material and widespread in both prokaryotes and eukaryotes， which exhibits multiple functions such as resisting oxidation and transferring electron in the electron transport chain[1]. Moreover， a lot of researches have been suggested that CoQ10is effective to treatment many diseases. Jahangard and coworkers[2]suggested that CoQ10might be considered a safe and effective strategy for treatment of patients with bipolar disorder during their depressive phase. Chen and coworkers[3]described that CoQ10could serve as an AMPK activator and regulate the hepatic lipid metabolism to inhibit the abnormal accumulation of hepatic lipids and prevent nonalcoholic fatty liver disease progression. Moreover， CoQ10benefits the treatment for patients with fibromyalgia[4]， early chronic Peyronies disease[5]， metabolic syndrome[6]. Currently， CoQ10is widely used in food， comestic and pharmaceutical industry and the requirement of CoQ10is increasing. Microbial fermentation is one of most promising way to produce natural functional CoQ10. Among which， Rhodobacter sphaeroides is proven to be an excellent microorganism for producing natural functional CoQ10[7]. In the present study， we optimized the production procedure for CoQ10production from the genetic engineered R. sphaeroides overexpressing UbiG， which is an oxygenmethyltransferase， participating in two steps for the synthesis of CoQ10in R. sphaeroides. UbiG first catalyzes 2decaprenyl6hydroxyphenol into 2decaprenyl6methoxyphenol， and catalyzes the 2decaprenyl3methyl5hydroxy6methoxy1，4benzoquinone into CoQ10， which is the last step for the formation of CoQ10in R. Sphaeroides[8]. Materials and Methods
Bacterial strain growth conditions
R. sphaeroides strains were grown at 30 ℃ in malate minimal medium in dark[9]. Growth under microaerobic conditions in the dark was performed as described in our previous study[10]. 1.5μg/ml tetracycline was used for cultivation of genetic engineered R. sphaeroides overexpressing UbiG.
Effects of inoculate amount on crude CoQ10production from genetic engineered R. sphaeroides strain
A single colony was inoculated into a 50ml flask containing 40 ml of malate minimal medium and grown under microaerobic growth conditions in dark at 30 ℃ until OD660reached 0.6. Then， precultures were inoculated into 100ml flasks containing 80 ml of malate minimal medium at the ratio of 1%， 2%， 3%， 4% and 5%， respectively. The cultures were grown under microaerobic conditions in dark at 30 ℃ for 48 h. Crude CoQ10was then respectively extracted from the cell cultures and quantified as described in our previous study[11]. The experiment was repeated three times.
Effects of incubation temperature on crude CoQ10production from genetic engineered R. sphaeroides strain
A single colony was inoculated into a 50ml flask containing 40 ml of malate minimal medium and grown under microaerobic conditions in dark at 30 ℃ until OD660reached 0.6. Then， the precultures were inoculated into 100ml flasks containing 80 ml of malate minimal medium at the ratio of 1∶ 50 and grown at microaerobic conditions in dark at 27， 30 and 33 ℃ for 48 h. CoQ10was respectively extracted from the cell cultures and quantified as described in our previous study[11]. The experiment was repeated three times.
Effects of fermentation duration on crude CoQ10production from genetic engineered R. sphaeroides strain
A single colony was inoculated into a 50ml flask containing 40 ml of malate minimal medium and grown under microaerobic conditions in dark at 30 ℃ until OD660reached 0.6. Then， precultures were respectively inoculated into 100ml flasks containing 80 ml of malate minimal medium at the ratio of 1∶ 50 and grown microaerobic conditions in dark at 30 ℃ for 36， 48， 60， 72 and 84 h.CoQ10was respectively extracted from the cell cultures and quantified as described in our previous study[11]. The experiment was repeated three times.
Effects of oxygen tension （fermentation medium volume to total flask volume） on crude CoQ10production from genetic engineered R. sphaeroides strain A single colony was inoculated into a 50ml flask containing 40 ml of malate minimal medium and grown under microaerobic conditions in dark at 30 ℃ until OD660reached 0.6. Then， precultures were respectively inoculated into five 100ml flasks containing 50， 60， 70， 80 and 90 ml of malate minimal medium at the ratio of 1∶ 50and grown under microaerobic conditions in dark at 30 ℃ for 48 h.Oxygen tension in the different shaken flasks with different volumes of medium was different caused by the steady rotation speed. CoQ10was extracted from the cell cultures and quantified as mentioned above， respectively. The experiment was repeated three times.
Results and Discussion
Optimization of fermentation time
The optimized fermentation time for the enhancement of crude CoQ10from genetic engineered strain R. sphaeroides overexpressing UbiG was tested， as observed in Fig. 1. The production of CoQ10was increased with the increase of fermentation duration from 24 to 48 h. The production of crude CoQ10extracted from the strain fermented for 24， 36 and 48 h， was 21.96， 26.99 and 40.49 mg/L， respectively. Compared with the production of crude CoQ10from the cell cultures fermented for 24 and 36 h， the production of crude CoQ10from the cell cultures incubated for 48 h was increased by 84.40% and 33.36%， respectively. However， the production of crude CoQ10was declined when fermented for more than 48 h. The production of crude CoQ10from the cell cultures incubated for 60 and 72 h was 21.17 and 13.64 mg/L， respectively， which were decreased by 47.71% and 66.33% compared with the CoQ10production extracted from the cell cultures incubated for 48 h， respectively. It is reasonable to speculate that fermentation duration for 48 h was the optimized fermentation duration， which was similar to optimized incubation during for the genetic engineered R. sphaeroides overexpressing UbiE as described in our previous study[10].
Optimization of inoculate amount
The experimental results for optimizing the inoculate amount for enhancement of crude CoQ10from genetic engineered R. sphaeroides overexpressing UbiG is revealed in Fig. 2. Obviously， the inoculate amount greatly affected the crude CoQ10production from genetic engineering R. sphaeroides overexpressing UbiG. The productionof crude CoQ10was 18.14， 39.17， 29.31， 22.75 and 24.56 mg/L when the inoculate amount was 1%， 2%， 4%， 6% and 8%， respectively. The production of crude CoQ10was highest when the inoculate amount was 2%， which was over 2 times of the crude CoQ10production when the inoculate amount was 1%. The crude CoQ10production was decreased by 25.17%， 41.93% and 37.29% when the inoculate amount was 4%， 6% and 8%， respectively compared with the crude CoQ10production at the inoculate amount of 2%. Optimization of fermentation temperature
The fermentation temperature was optimized for enhancement of crude CoQ10production from the genetic engineered strain R. sphaeroides overexpressing UbiG， as seen in Fig. 3. It could be seen that from 23 to 30 ℃ with the increase of the incubation temperature， the production of crude CoQ10from the genetic engineered R. sphaeroides overexpressing UbiG was increased. However， with the increase of incubation temperature from 30 to 37 ℃， the production of crude CoQ10from the genetic engineered R. sphaeroides overexpressing UbiG was decreased. It was similar with the genetic engineered R. sphaeroides overexpressing UbiE[10]. Clearly， 30 ℃ was the optimized temperature for production of crude CoQ10from the genetic engineered strain， with the yield of 39.17 mg/L. Compared with the crude CoQ10production at 23 and 27 ℃， the CoQ10production at 30 ℃ was increased by 509.36% and 27.93%， respectively. While， the production of CoQ10production at 33 and 37 ℃ was decreased by 39.88% and 50.66%， respectively.
Agricultural Biotechnology 2020
Optimization of fermentation medium volume used in shaken flasks （changes dissolved oxygen tension）
The dissolved oxygen tension will be affected by filling different volumes of medium in the same volume flasks when shaken at steady speed. The ubiG gene is initiated by the puf operon promoter which is oxygen tension dependent[12]. When the dissolved oxygen tension is high， the PpsR repressor will be bound to the puf promoter and overexpression of ubiG will be repressed. However， at low dissolved oxygen tension， the PpsR repressor will be bound with the antirepressor protein AppA and form a complex， and thus the PpsR repressor cannot bind the puf promoter[13]. Consequently， the overexpression of ubiG will be activated. Effect of oxygen tension on the production of crude CoQ10is shown in Fig. 4. The highest production of CoQ10was obtained when the medium volume to whole flask volume was 80%， suggesting the optimized oxygen tension was obtained for growth and fermentation. The production of crude CoQ10was 11.02， 18.85， 26.62， 37.21 and 31.63mg/L， respectively when the filled medium volume was 50%， 60%， 70%， 80% and 90% of the total flask volume. When the volumes were lower than 80%， the dissolved oxygen tension in the medium was relatively higher and thus the puf promoter activity was repressed. On the other hand， the bacteria need oxygen for survival， so when the medium volume was 90%， the production of crude CoQ10was lower than that of 80%. It was concluded that dissolved oxygen tension was crucial for the production of crude CoQ10from the genetic engineering strains. Conclusion
The optimal processes for producing CoQ10from genetic engineered R. sphaeroides overexpressing UbiG were： fermentation duration 48 h， inoculate amount 2%， fermentation temperature 30 ℃， and volume of fermentation medium to flask total volume 80%.
References
[1] RIZVI A， CHIBBER S， NASEEM I. Cu（II）vitamin D interaction leads to free radicalmediumted cellular DNA damage： a novel putative mechanism for its selective cytotoxic action against malignant cells[J]. Tumour Biol， 2015， 36（3）： 1695-1700.
[2] JAHANGARD L， YASREBIFAR F， HAGHIGHI M， et al. Influence of adjuvant Coenzyme Q10on inflammatory and oxidative stress biomarkers in patients with bipolar disorders during the depressive episode[J]. Molecular Biology Reports， 2019， 46（5）： 5333-5343.
[3] CHEN K， CHEN X， XUE H， et al. Coenzyme Q10attenuates highfat dietinduced nonalcoholic fatty liver disease through activation of the AMPK pathway[J]. Food Funct， 2019， 10（2）： 814-823.
[4] CORDERO MD， MORENOFERNANDEZ AM， DEMIGUEL M， et al. Coenzyme Q10distribution in blood is altered in patients with fibromyalgia[J]. Clin Biochem， 2009， 42（7-8）： 732-735.
[5] SAFARINEJAD MR. Safety and efficacy of coenzyme Q10supplementation in early chronic Peyronies disease： a doubleblind， placebocontrolled randomized study[J]. Int J Impot Res， 2010， 22（5）： 298-309.
[6] RAYGAN F， REZAVANDI Z， DADKHAH TEHRANI S， et al. The effects of coenzyme Q10administration on glucose homeostasis parameters， lipid profiles， biomarkers of inflammation and oxidative stress in patients with metabolic syndrome[J]. Eur J Nutr， 2016， 55（8）： 2357-2364.
[7] LU W， SHI Y， HE S， et al. Enhanced production of CoQ10by constitutive overexpression of 3demethyl ubiquinone9 3methyltransferase under tac promoter in Rhodobacter sphaeroides[J]. Biochemical Engineering Journal， 2017（72）： 42-47.
[8] LU W， YE L， LV X， et al. Identification and elimination of metabolic bottlenecks in the quinone modification pathway for enhanced coenzyme Q10production in Rhodobacter sphaeroides[J]. Metab Eng， 2015（29）： 208-216.
[9] REMES B， BERGHOFF BA， FORSTNER KU， et al. Role of oxygen and the OxyR protein in the response to iron limitation in Rhodobacter sphaeroides[J]. BMC Genomics， 2014（15）： 794.
[10] TANG K， ZHAO ZZ， ZHANG L， et al. Optimization of coenzyme Q10production procedure from Rhodobacter sphaeroides overexpressing UbiE[J]. Agricultural Biotechnology， 2019， 8（5）： 19-20， 25.
[11] TANG K， WANG J， WANG W， et al. Production of functional coenzyme Q10from genetic engineered Rhodobacter sphaeroides[J]. Advance Journal of Food Science and Technology， 2019， 17（3）： 48-53.
[12] BOWMAN WC， DU S， BAUER CE， et al. In vitro activation and repression of photosynthesis gene transcription in Rhodobacter capsulatus[J]. Mol Microbiol， 1999， 33（2）： 429-437.
[13] MASUDA S， BAUER CE. AppA is a blue light photoreceptor that antirepresses photosynthesis gene expression in Rhodobacter sphaeroides[J]. Cell， 2002， 110（5）： 613-623.