The electronic structure of protein chains L and M in photosynthetic reaction center (PRC) of Rhodobacter sphaeroides (Van Niel) Imhoff, Truper et Pfennig) was studied by using the Overlapping Dimer Approximation meth...The electronic structure of protein chains L and M in photosynthetic reaction center (PRC) of Rhodobacter sphaeroides (Van Niel) Imhoff, Truper et Pfennig) was studied by using the Overlapping Dimer Approximation method and the Extended Negative Factor Counter method at ab initio level. The result indicated that: (1) Amino acid residues, the molecular orbitals of which composed the main components of frontier orbitals of protein chain L (M), are located at the random coil areas of chain L (alpha helix areas of chain M). Since the random coil is flexible and more easy to change its conformation in the electron transfer process and to reduce the energy of the system, and the structure of the alpha helix is reletively stable, this difference might be one of the causes for the electron transfer in photosynthetic reaction center (PRC) only takes place along the L branch. (2) The His residues which axially coordinated to the 'special pair' P and accessory chlorophyll molecules (ABChls) are essentially important for the E-LUMO levels of P and ABChl. But, the corresponding molecular orbitals of these His residues do not appear in the composition of frontier orbitals of protein chains. It means that the interaction between pigment molecules and protein chains do not influence the contribution to the frontier orbitals of protein chains explicitly, but influences the corresponding E-LUMO levels significantly.展开更多
Coenzyme Q10 is widely used in food,cosmetics and pharmaceuticals,possessing a broad market.Rhodobacter sphaeroides is enriched in natural coenzyme Q10 and is becoming an important microorganism for producing natural ...Coenzyme Q10 is widely used in food,cosmetics and pharmaceuticals,possessing a broad market.Rhodobacter sphaeroides is enriched in natural coenzyme Q10 and is becoming an important microorganism for producing natural coenzyme Q10.The paper reviewed the biosynthesis pathways of coenzyme Q10 in R.sphaeroides and the advances in enhancement of coenzyme Q10 production in R.sphaeroides based on metabolic engineering.展开更多
In the present study,single factors including inoculation amount,fermentation temperature,fermentation duration and ratio of fermentation medium volume to total flask volume were optimized for enhancing the production...In the present study,single factors including inoculation amount,fermentation temperature,fermentation duration and ratio of fermentation medium volume to total flask volume were optimized for enhancing the production of coenzyme Q10 from genetic engineered Rhodobacter sphaeroides overexpressing UbiE.The experimental results suggested that optimal single factors were: inoculation amount 2%,fermentation temperature 30 ℃,fermentation duration 48 h,and fermentation medium volume to total flask volume 80%.The present study will promote the large scale production of CoQ10 from microorganisms.展开更多
Carotenoids act as precursors of vitamin A,antioxidants,enhancers of immunity,and are thus widely used in food and pharmaceutical industry.Microbial fermentation is one of the most important solutions for production o...Carotenoids act as precursors of vitamin A,antioxidants,enhancers of immunity,and are thus widely used in food and pharmaceutical industry.Microbial fermentation is one of the most important solutions for production of natural carotenoids.Rhodobacter sphaeroides is one of most promising bacteria employed for large scale production of carotenoids.In the present study,crtA located in the carotenoids biosynthesis pathway in R.sphaeroides was amplified by PCR.The overexpression vector pRKcrtA was constructed and subsequently transferred into R.sphaeroides,producing the genetically engineered strain R.sphaeroides 2.4.1/pRKcrtA overexpressing crtA.The carotenoid production from the genetically engineered strain was significantly increased.Fermentation procedure was optimized for further enhanced carotenoids production.展开更多
Rhodobacter sphaeroides is a purple non-sulfur bacterium that belongs to the α-3 subdivision of Proteobacteria. R. sphaeroides is a model bacterial species because of its complex genome structure and expanded metabol...Rhodobacter sphaeroides is a purple non-sulfur bacterium that belongs to the α-3 subdivision of Proteobacteria. R. sphaeroides is a model bacterial species because of its complex genome structure and expanded metabolic capabilities. The genome of R. sphaeroides consists of two circular chromosomes and five endogenous plasmids. It has the ability to grow under a wide variety of environmental conditions. It grows aerobically (~20% O2), semi-aerobically (~2% O2), and photosynthetically (under anaerobic condition plus light). It has been previously shown that many bacterial species utilize a number of alternate carbon sources for their optimal growth under a variety of growth conditions. We hypothesize that different or an additional carbon source in the minimal medium differentially affects the bacterial growth under dark-aerobic conditions. The bacterial growth kinetics and the number of cells in the bacterial culture were analyzed by measuring the optical density (OD at 600 nm) and the colony forming units (CFUs) at regular intervals of bacterial cultures. Results reveal that sodium succinate is the preferred sole carbon source for the optimal growth of R. sphaeroides. The results of growth kinetics and CFUs together concluded that from the tested carbon sources, sodium succinate is the best single carbon source in the minimal media for the optimal growth of R. sphaeroides. Interestingly, cell culture grown in SIS supplemented with sodium acetate exhibits a prolonged lag phase with the lowest ODs and CFUs that later switches to the growth-burst phase support previously discovered similar phenomenon of the growth-rate switch in the presence of acetate metabolism. Future work will utilize the aerobically grown R. sphaeroides’ cells as a biocatalyst to deplete the oxygen levels from natural gas streams and industrial gas pipelines.展开更多
In order to enhance the degrading protein capability of purple non-sulfur bacteria(PNSB),an effective strain,L2,was used to co-culture with Rhodobacter sphaeroides ATCC17023.The effects of added strain on protein remo...In order to enhance the degrading protein capability of purple non-sulfur bacteria(PNSB),an effective strain,L2,was used to co-culture with Rhodobacter sphaeroides ATCC17023.The effects of added strain on protein removal of R.sphaeroides were investigated.Results showed that strain L2,being identified as Bacillus thuringiensis/cereus,had a high potential for producing protease with a production of 295 U/m L.The optimal B.thuringiensis/cereus(40 μL) could significantly increase protein degradation of R.sphaeroides.Protein removal and biomass production were improved by 483% and 67%,respectively.R.sphaeroides/total biomass production was more than 95%.Theoretical analysis revealed that R.sphaeroides syntrophically interacted with B.thuringiensis/cereus.Protein degradation of B.thuringiensis/cereus provided small molecule substrates(VFAs) for R.sphaeroides growth and cells materials synthesis.展开更多
Rhodobacter sphaeroides RV (RV) produces high yields of hydrogen from organic acids in the presence of light. The hydrogen production from acetate is lower than that from lactate, probably because of its low ability t...Rhodobacter sphaeroides RV (RV) produces high yields of hydrogen from organic acids in the presence of light. The hydrogen production from acetate is lower than that from lactate, probably because of its low ability to metabolize acetate. In this study, gene of acetaldehyde dehydrogenase (ACDH, EC 1.2.1.10) that catalyzes the reversible conversion of acetaldehyde and CoA to acetyl-CoA with the concurrent reduction of NAD to NADH, is overexpressed in the RV strain. The produced acetyl-CoA can be oxidized to carbon dioxide in the tricarboxylic acid (TCA) cycle, wherein electrons are generated and used for hydrogen production. The byproduct NADH can be used as reducing agent for acetate to produce acetaldehyde by acetate dehydrogenase. The recombinant RV strain (RVAC) expressing the ACDH gene showed ACDH activity with a specific activity of 3.2 mU/ mg, and the RV and the recombinant RV strain that harbored the intact (empty) plasmid pLP-1.2 (RVI) showed no detectable ACDH activity. The hydrogen yields of the RVAC strain from 21-mM acetate were 1.5-fold higher than that of the wild type RV strain and also that of the RVI strain. In contrast, hydrogen yield from 21-mM lactate was 30% lower than that in the control strains.展开更多
The optimization of fermentation medium is important for synthetic biological secondary metabolite productions.The effect of rotation speed,inoculum amount,and medium supplements on the cell growth and Lycogen^TM secr...The optimization of fermentation medium is important for synthetic biological secondary metabolite productions.The effect of rotation speed,inoculum amount,and medium supplements on the cell growth and Lycogen^TM secretion of photobacterium Rhodobacter sphaeroides WL-APD911 was evaluated.The results reveal that a higher rotational speed exhibit a higher cell density,and the increasing in the amount of inoculum amount show a slight augment on the growth of R.sphaeroides WL-APD911.In the case of nitrogen sources adding,Lycogen^TM production was achieved with a 0.5 mM l-lysine supplementation.Moreover,the attention of Tween 80 presented a tremendous increase in the secondary metabolite.Response surface methodology(RSM)exhibited the optimization of medium supplements for Lycogen^TM invention is accomplished at molasses concentration of 10 g/L,yeast extract concentration of 40 g/L,0.3%Tween 80 and NaCl concentration of 5 g/L,respectively.Further,the batch fermentation is carried out in both 5 L and 20 L fermentors to study the scale-up process factors to be adopted.At a 20 L fermentor,Lycogen^TM yields under the optimal culture condition are over 2 times than in the shake flask.The present results provide the Lycogen^TM optimal culture mediums,scale-up procedures and efficient extractions from R.sphaeroides WL-APD911.展开更多
The bacterial cell cycle consists of a series of genetically coordinated biochemical and biophysical events. In Caulobacter crescentus, CtrA is an essential cell cycle regulator that modulates many cell cycle processe...The bacterial cell cycle consists of a series of genetically coordinated biochemical and biophysical events. In Caulobacter crescentus, CtrA is an essential cell cycle regulator that modulates many cell cycle processes. In the present study, the role of the CtrA was investigated in Rhodobacter sphaeroides 2.4.1 by employing genetic, molecular, and bioinformatic approaches. Examination of the ctrA-null mutant revealed that the loss of CtrA did not affect growth characteristics and cell morphology in R. sphaeroides when grown under aerobic or photosynthetic growth conditions but slower growth was noticed in the anaerobic-dark-DMSO condition. Phylogenetic analyses demonstrated that CtrA has diversified its role in major lineages of α-Proteobacteria and has possibly been involved in adaptation to variable lifestyles. Analysis of the CtrA binding sites in the R. sphaeroides genome suggests that CtrA may regulate 127 genes involving different cellular processes. Protein homology searches revealed that only a small number of ctrA-regulated genes are homologous across C. crescentus, R. capsulatus, and R. sphaeroides. Comparison of the functions of putative ctrA-regulated genes in C. crescentus, R. capsulatus, and R. sphaeroides revealed that all three species possessed broad pathway control across a variety of cluster of orthologous gene functions (COGs). However, interestingly, it seems that the essentiality of CtrA in C. crescentus may depend more on the selective control that it exerts on a few critical cell cycle genes and pathways that are not controlled by CtrA in a similar fashion in R. capsulatus and R. sphaeroides.展开更多
Heavy metal pollution is a worldwide problem with many associated health risks, including bone loss, kidney damage, and several forms of cancer. There is a great need of bioremediation of these toxic metals from the e...Heavy metal pollution is a worldwide problem with many associated health risks, including bone loss, kidney damage, and several forms of cancer. There is a great need of bioremediation of these toxic metals from the environment, as well as implementing a monitoring system to control the spreading pollution. This study focuses on the bioremediation potential of Rhodobacter sphaeroides in the presence of the toxic gold chloride (AuCl3). Growth characteristics of the bacterial cells exposed to a range of toxic gold concentrations were analyzed through the growth kinetics and the colony forming units under aerobic, photosynthetic, and anaerobic growth conditions. The localization of the gold particles within two cellular fractions, cytoplasm and the plasma membrane, are analyzed using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). Results of this study demonstrated the photosynthetic growth condition as best suited for the metal tolerance, compared to the aerobic and anaerobic growth conditions. Results also revealed the overall accumulation and localization of gold particles, while not different between the membrane and the cytoplasmic fractions increased at different concentrations of the gold contamination. The results of the localization under photosynthetic growth condition revealed the accumulation reached the highest very quickly, and an overall shift in localization of the gold particles from an equal distribution to an increase within the membrane fraction at the highest concentrations of gold contamination. The localization of the gold particles was validated by Transmission Electron Microscopy (TEM) where the results confirmed the increase in accumulation within the membrane, and photosynthetic membranes, of R. sphaeroides.展开更多
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 ...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 Q10 from 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 CoQ10 from microorganisms.展开更多
The conventional treatment method of soybean wastewater is expensive and generates waste sludge that requires further handling.Purple nonsulfur bacteria(PNSB)wastewater treatment is a clean technology and can generate...The conventional treatment method of soybean wastewater is expensive and generates waste sludge that requires further handling.Purple nonsulfur bacteria(PNSB)wastewater treatment is a clean technology and can generate single cell protein while degrading pollutants.A wild strain of PNSB,Rhodobacter sphaeroides Z08,was isolated from local soil and was used to treat soybean wastewater.To develop a cost-effective process,the work was performed under natural conditions without artificial light,aeration,nutrients addition,or pH and temperature adjustment.The results showed that the wild strain Rhodobacter sphaeroides Z08 could grow well under natural conditions.The growth curve showed two quickgrowth periods and a turning point.The Z08 treatment of soybean wastewater was zero order reaction and COD reduction was 96%after 10 d.The major byproducts of the process were C2-C5 organic acids,predominantly butyric acid.No alcohol was found in the effluent.The initial COD/bacterial-mass ratio(F/M)had a significant effect on soybean wastewater treatment efficiency.When the initial F/M was lower than 10 mg-COD/mg-bacteria,a sufficient amount of time to achieve 90%of COD reduction was only three days.The Z08 biomass yield was 0.28 g·g^(–1),and the bacterial protein content was 52%.展开更多
The Rhodobacter capsulatus hemA gene, which encodes 5-aminolevulinic acid synthase （ALAS）, was expressed in Escherichia coil Rosetta （DE3） and the enzymatic properties of the purified recombinant ALAS （RC-ALAS） ...The Rhodobacter capsulatus hemA gene, which encodes 5-aminolevulinic acid synthase （ALAS）, was expressed in Escherichia coil Rosetta （DE3） and the enzymatic properties of the purified recombinant ALAS （RC-ALAS） were studied. Compared with ALASs encoded by hemA genes from Agrobacterium radiobacter（AR-ALAS） and Rhodobacter sphaeroides （RS-ALAS）, the specific activity of RC-ALAS reached 198.2 U/mg, which was about 31.2% and 69.5% higher than those of AR-ALAS （151.1 U/mg） and RS-ALAS （116.9 U/mg）, respectively. The optimum pH values and temperatures of the three above mentioned enzymes were all pH 7.5 and 37 ℃, respectively. Moreover, RC-ALAS was more sensitive to pH, while the other two were sensitive to temperature. The effects of metals, ethylene diamine tetraacetic acid （EDTA）, and sodium dodecyl sulfate （SDS） on the three ALASs were also investigated. The results indicate that they had the same effects on the activities of the three ALASs. SDS and metal ions such as Co^2＋, Zn^2＋, and Cu^2＋ strongly inhibited the activities of the ALASs, while Mn^2＋ exerted slight inhibition, and K^＋, Ca^2＋, Ba^2＋, Mg^2＋, or EDTA had no significant effect. The specificity constant of succinyl coenzyme A [（kcatlKm）^S-CoA] of RC-ALAS was 1.4989, which was higher than those of AR-ALAS （0.7456） and RS-ALAS （1.1699）, showing its high catalytic efficiency. The fed-batch fermentation was conducted using the recombinant strain containing the R. capsulatus hemA gene, and the yield of 5-aminolevulinic acid （ALA） achieved was 8.8 g/L （67 mmol/L） under the appropriate conditions.展开更多
The versatile photosyntheticα-proteobacterium Rhodobacter sphaeroides,has recently been extensively engineered as a novel microbial cell factory(MCF)to produce pharmaceuticals,nutraceuticals,commodity chemicals and e...The versatile photosyntheticα-proteobacterium Rhodobacter sphaeroides,has recently been extensively engineered as a novel microbial cell factory(MCF)to produce pharmaceuticals,nutraceuticals,commodity chemicals and even hydrogen.However,there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R.sphaeroides.In this study,several native promoters from R.sphaeroides JDW-710(JDW-710),an industrial strain producing high levels of co-enzyme Q10(Q10)were selected on the basis of transcriptomic analysis.These candidate promoters were then characterized by using gusA as a reporter gene.Two native promoters,Prsp_7571 and Prsp_6124,showed 620%and 800%higher activity,respectively,than the tac promoter,which has previously been used for gene overexpression in R.sphaeroides.In addition,a Prsp_7571-derived synthetic promoter library with strengths ranging from 54%to 3200%of that of the tac promoter,was created on the basis of visualization of red fluorescent protein(RFP)expression in R.sphaeroides.Finally,as a demonstration,the synthetic pathway of Q10 was modulated by the selected promoter T334*in JDW-710;the Q10 yield in shake-flasks increased 28%and the production reached 226 mg/L.These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R.sphaeroides-derived MCFs.展开更多
文摘The electronic structure of protein chains L and M in photosynthetic reaction center (PRC) of Rhodobacter sphaeroides (Van Niel) Imhoff, Truper et Pfennig) was studied by using the Overlapping Dimer Approximation method and the Extended Negative Factor Counter method at ab initio level. The result indicated that: (1) Amino acid residues, the molecular orbitals of which composed the main components of frontier orbitals of protein chain L (M), are located at the random coil areas of chain L (alpha helix areas of chain M). Since the random coil is flexible and more easy to change its conformation in the electron transfer process and to reduce the energy of the system, and the structure of the alpha helix is reletively stable, this difference might be one of the causes for the electron transfer in photosynthetic reaction center (PRC) only takes place along the L branch. (2) The His residues which axially coordinated to the 'special pair' P and accessory chlorophyll molecules (ABChls) are essentially important for the E-LUMO levels of P and ABChl. But, the corresponding molecular orbitals of these His residues do not appear in the composition of frontier orbitals of protein chains. It means that the interaction between pigment molecules and protein chains do not influence the contribution to the frontier orbitals of protein chains explicitly, but influences the corresponding E-LUMO levels significantly.
基金Supported by Talent Project of Sichuan University of Science&Engineering(2015RC27)Meat Processing Key Laboratory of Sichuan Province(16R-27)
文摘Coenzyme Q10 is widely used in food,cosmetics and pharmaceuticals,possessing a broad market.Rhodobacter sphaeroides is enriched in natural coenzyme Q10 and is becoming an important microorganism for producing natural coenzyme Q10.The paper reviewed the biosynthesis pathways of coenzyme Q10 in R.sphaeroides and the advances in enhancement of coenzyme Q10 production in R.sphaeroides based on metabolic engineering.
基金Supported by the Research Project of Sichuan University of Science&Engineering(2015RC27)Scientific Research Foundation of the Education Department of Sichuan Province(15ZA0222)Research Project of Meat Processing Key Laboratory of Sichuan Province(16R-27)
文摘In the present study,single factors including inoculation amount,fermentation temperature,fermentation duration and ratio of fermentation medium volume to total flask volume were optimized for enhancing the production of coenzyme Q10 from genetic engineered Rhodobacter sphaeroides overexpressing UbiE.The experimental results suggested that optimal single factors were: inoculation amount 2%,fermentation temperature 30 ℃,fermentation duration 48 h,and fermentation medium volume to total flask volume 80%.The present study will promote the large scale production of CoQ10 from microorganisms.
基金Supported by the Project of Sichuan Science and Technology Department(2019YJ0673)National Modern Agriculture Industry System/Sichuan Live Pig Innovation Team(SCSZTD-3-007)。
文摘Carotenoids act as precursors of vitamin A,antioxidants,enhancers of immunity,and are thus widely used in food and pharmaceutical industry.Microbial fermentation is one of the most important solutions for production of natural carotenoids.Rhodobacter sphaeroides is one of most promising bacteria employed for large scale production of carotenoids.In the present study,crtA located in the carotenoids biosynthesis pathway in R.sphaeroides was amplified by PCR.The overexpression vector pRKcrtA was constructed and subsequently transferred into R.sphaeroides,producing the genetically engineered strain R.sphaeroides 2.4.1/pRKcrtA overexpressing crtA.The carotenoid production from the genetically engineered strain was significantly increased.Fermentation procedure was optimized for further enhanced carotenoids production.
文摘Rhodobacter sphaeroides is a purple non-sulfur bacterium that belongs to the α-3 subdivision of Proteobacteria. R. sphaeroides is a model bacterial species because of its complex genome structure and expanded metabolic capabilities. The genome of R. sphaeroides consists of two circular chromosomes and five endogenous plasmids. It has the ability to grow under a wide variety of environmental conditions. It grows aerobically (~20% O2), semi-aerobically (~2% O2), and photosynthetically (under anaerobic condition plus light). It has been previously shown that many bacterial species utilize a number of alternate carbon sources for their optimal growth under a variety of growth conditions. We hypothesize that different or an additional carbon source in the minimal medium differentially affects the bacterial growth under dark-aerobic conditions. The bacterial growth kinetics and the number of cells in the bacterial culture were analyzed by measuring the optical density (OD at 600 nm) and the colony forming units (CFUs) at regular intervals of bacterial cultures. Results reveal that sodium succinate is the preferred sole carbon source for the optimal growth of R. sphaeroides. The results of growth kinetics and CFUs together concluded that from the tested carbon sources, sodium succinate is the best single carbon source in the minimal media for the optimal growth of R. sphaeroides. Interestingly, cell culture grown in SIS supplemented with sodium acetate exhibits a prolonged lag phase with the lowest ODs and CFUs that later switches to the growth-burst phase support previously discovered similar phenomenon of the growth-rate switch in the presence of acetate metabolism. Future work will utilize the aerobically grown R. sphaeroides’ cells as a biocatalyst to deplete the oxygen levels from natural gas streams and industrial gas pipelines.
基金Sponsored by the National Natural Science Foundation of China(Grant No.51278489)
文摘In order to enhance the degrading protein capability of purple non-sulfur bacteria(PNSB),an effective strain,L2,was used to co-culture with Rhodobacter sphaeroides ATCC17023.The effects of added strain on protein removal of R.sphaeroides were investigated.Results showed that strain L2,being identified as Bacillus thuringiensis/cereus,had a high potential for producing protease with a production of 295 U/m L.The optimal B.thuringiensis/cereus(40 μL) could significantly increase protein degradation of R.sphaeroides.Protein removal and biomass production were improved by 483% and 67%,respectively.R.sphaeroides/total biomass production was more than 95%.Theoretical analysis revealed that R.sphaeroides syntrophically interacted with B.thuringiensis/cereus.Protein degradation of B.thuringiensis/cereus provided small molecule substrates(VFAs) for R.sphaeroides growth and cells materials synthesis.
文摘Rhodobacter sphaeroides RV (RV) produces high yields of hydrogen from organic acids in the presence of light. The hydrogen production from acetate is lower than that from lactate, probably because of its low ability to metabolize acetate. In this study, gene of acetaldehyde dehydrogenase (ACDH, EC 1.2.1.10) that catalyzes the reversible conversion of acetaldehyde and CoA to acetyl-CoA with the concurrent reduction of NAD to NADH, is overexpressed in the RV strain. The produced acetyl-CoA can be oxidized to carbon dioxide in the tricarboxylic acid (TCA) cycle, wherein electrons are generated and used for hydrogen production. The byproduct NADH can be used as reducing agent for acetate to produce acetaldehyde by acetate dehydrogenase. The recombinant RV strain (RVAC) expressing the ACDH gene showed ACDH activity with a specific activity of 3.2 mU/ mg, and the RV and the recombinant RV strain that harbored the intact (empty) plasmid pLP-1.2 (RVI) showed no detectable ACDH activity. The hydrogen yields of the RVAC strain from 21-mM acetate were 1.5-fold higher than that of the wild type RV strain and also that of the RVI strain. In contrast, hydrogen yield from 21-mM lactate was 30% lower than that in the control strains.
基金This work was supported by grants from the Ministry of Science and Technology,Taiwan,ROC(MOST 104-2221-E-005-096-MY2,and MOST 104-2628-E-005-004-MY3).We thank the projects of Center for Stem Cell Research,Kaohsiung Medical University,Kaohsiung,Taiwan,KMU-TP104G00 and KMU-TP104G02-05.The financial supports were also from KMU-DK105005 and NSYSUKMU105-P 007.
文摘The optimization of fermentation medium is important for synthetic biological secondary metabolite productions.The effect of rotation speed,inoculum amount,and medium supplements on the cell growth and Lycogen^TM secretion of photobacterium Rhodobacter sphaeroides WL-APD911 was evaluated.The results reveal that a higher rotational speed exhibit a higher cell density,and the increasing in the amount of inoculum amount show a slight augment on the growth of R.sphaeroides WL-APD911.In the case of nitrogen sources adding,Lycogen^TM production was achieved with a 0.5 mM l-lysine supplementation.Moreover,the attention of Tween 80 presented a tremendous increase in the secondary metabolite.Response surface methodology(RSM)exhibited the optimization of medium supplements for Lycogen^TM invention is accomplished at molasses concentration of 10 g/L,yeast extract concentration of 40 g/L,0.3%Tween 80 and NaCl concentration of 5 g/L,respectively.Further,the batch fermentation is carried out in both 5 L and 20 L fermentors to study the scale-up process factors to be adopted.At a 20 L fermentor,Lycogen^TM yields under the optimal culture condition are over 2 times than in the shake flask.The present results provide the Lycogen^TM optimal culture mediums,scale-up procedures and efficient extractions from R.sphaeroides WL-APD911.
文摘The bacterial cell cycle consists of a series of genetically coordinated biochemical and biophysical events. In Caulobacter crescentus, CtrA is an essential cell cycle regulator that modulates many cell cycle processes. In the present study, the role of the CtrA was investigated in Rhodobacter sphaeroides 2.4.1 by employing genetic, molecular, and bioinformatic approaches. Examination of the ctrA-null mutant revealed that the loss of CtrA did not affect growth characteristics and cell morphology in R. sphaeroides when grown under aerobic or photosynthetic growth conditions but slower growth was noticed in the anaerobic-dark-DMSO condition. Phylogenetic analyses demonstrated that CtrA has diversified its role in major lineages of α-Proteobacteria and has possibly been involved in adaptation to variable lifestyles. Analysis of the CtrA binding sites in the R. sphaeroides genome suggests that CtrA may regulate 127 genes involving different cellular processes. Protein homology searches revealed that only a small number of ctrA-regulated genes are homologous across C. crescentus, R. capsulatus, and R. sphaeroides. Comparison of the functions of putative ctrA-regulated genes in C. crescentus, R. capsulatus, and R. sphaeroides revealed that all three species possessed broad pathway control across a variety of cluster of orthologous gene functions (COGs). However, interestingly, it seems that the essentiality of CtrA in C. crescentus may depend more on the selective control that it exerts on a few critical cell cycle genes and pathways that are not controlled by CtrA in a similar fashion in R. capsulatus and R. sphaeroides.
文摘Heavy metal pollution is a worldwide problem with many associated health risks, including bone loss, kidney damage, and several forms of cancer. There is a great need of bioremediation of these toxic metals from the environment, as well as implementing a monitoring system to control the spreading pollution. This study focuses on the bioremediation potential of Rhodobacter sphaeroides in the presence of the toxic gold chloride (AuCl3). Growth characteristics of the bacterial cells exposed to a range of toxic gold concentrations were analyzed through the growth kinetics and the colony forming units under aerobic, photosynthetic, and anaerobic growth conditions. The localization of the gold particles within two cellular fractions, cytoplasm and the plasma membrane, are analyzed using Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). Results of this study demonstrated the photosynthetic growth condition as best suited for the metal tolerance, compared to the aerobic and anaerobic growth conditions. Results also revealed the overall accumulation and localization of gold particles, while not different between the membrane and the cytoplasmic fractions increased at different concentrations of the gold contamination. The results of the localization under photosynthetic growth condition revealed the accumulation reached the highest very quickly, and an overall shift in localization of the gold particles from an equal distribution to an increase within the membrane fraction at the highest concentrations of gold contamination. The localization of the gold particles was validated by Transmission Electron Microscopy (TEM) where the results confirmed the increase in accumulation within the membrane, and photosynthetic membranes, of R. sphaeroides.
基金Supported by the Project of Sichuan Science and Technology Department(2019YJ0673)National Modern Agriculture Industry System/Sichuan Live Pig Innovation Team(SCSZTD-3-007)
文摘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 Q10 from 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 CoQ10 from microorganisms.
基金the Chinese Ministry of Science&Technology(No.2006BAC19B04)the National Natural Science Foundation of China(Grant No.50978072).
文摘The conventional treatment method of soybean wastewater is expensive and generates waste sludge that requires further handling.Purple nonsulfur bacteria(PNSB)wastewater treatment is a clean technology and can generate single cell protein while degrading pollutants.A wild strain of PNSB,Rhodobacter sphaeroides Z08,was isolated from local soil and was used to treat soybean wastewater.To develop a cost-effective process,the work was performed under natural conditions without artificial light,aeration,nutrients addition,or pH and temperature adjustment.The results showed that the wild strain Rhodobacter sphaeroides Z08 could grow well under natural conditions.The growth curve showed two quickgrowth periods and a turning point.The Z08 treatment of soybean wastewater was zero order reaction and COD reduction was 96%after 10 d.The major byproducts of the process were C2-C5 organic acids,predominantly butyric acid.No alcohol was found in the effluent.The initial COD/bacterial-mass ratio(F/M)had a significant effect on soybean wastewater treatment efficiency.When the initial F/M was lower than 10 mg-COD/mg-bacteria,a sufficient amount of time to achieve 90%of COD reduction was only three days.The Z08 biomass yield was 0.28 g·g^(–1),and the bacterial protein content was 52%.
基金Project supported by the National Natural Science Foundation of China (No.20306026)the National Basic Research Program (973) of China (No.2007CB707805)
文摘The Rhodobacter capsulatus hemA gene, which encodes 5-aminolevulinic acid synthase （ALAS）, was expressed in Escherichia coil Rosetta （DE3） and the enzymatic properties of the purified recombinant ALAS （RC-ALAS） were studied. Compared with ALASs encoded by hemA genes from Agrobacterium radiobacter（AR-ALAS） and Rhodobacter sphaeroides （RS-ALAS）, the specific activity of RC-ALAS reached 198.2 U/mg, which was about 31.2% and 69.5% higher than those of AR-ALAS （151.1 U/mg） and RS-ALAS （116.9 U/mg）, respectively. The optimum pH values and temperatures of the three above mentioned enzymes were all pH 7.5 and 37 ℃, respectively. Moreover, RC-ALAS was more sensitive to pH, while the other two were sensitive to temperature. The effects of metals, ethylene diamine tetraacetic acid （EDTA）, and sodium dodecyl sulfate （SDS） on the three ALASs were also investigated. The results indicate that they had the same effects on the activities of the three ALASs. SDS and metal ions such as Co^2＋, Zn^2＋, and Cu^2＋ strongly inhibited the activities of the ALASs, while Mn^2＋ exerted slight inhibition, and K^＋, Ca^2＋, Ba^2＋, Mg^2＋, or EDTA had no significant effect. The specificity constant of succinyl coenzyme A [（kcatlKm）^S-CoA] of RC-ALAS was 1.4989, which was higher than those of AR-ALAS （0.7456） and RS-ALAS （1.1699）, showing its high catalytic efficiency. The fed-batch fermentation was conducted using the recombinant strain containing the R. capsulatus hemA gene, and the yield of 5-aminolevulinic acid （ALA） achieved was 8.8 g/L （67 mmol/L） under the appropriate conditions.
基金This work was supported by the National Natural Science Foundation of China[31870040]the National Key Research and Development Project(2020YFA0907804,2020YFA0907304)+1 种基金the“111”Project of China[B18022]the Fundamental Research Funds for the Central Universities[22221818014],and the Open Project Funding of the State Key Laboratory of Bioreactor Engineering.
文摘The versatile photosyntheticα-proteobacterium Rhodobacter sphaeroides,has recently been extensively engineered as a novel microbial cell factory(MCF)to produce pharmaceuticals,nutraceuticals,commodity chemicals and even hydrogen.However,there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R.sphaeroides.In this study,several native promoters from R.sphaeroides JDW-710(JDW-710),an industrial strain producing high levels of co-enzyme Q10(Q10)were selected on the basis of transcriptomic analysis.These candidate promoters were then characterized by using gusA as a reporter gene.Two native promoters,Prsp_7571 and Prsp_6124,showed 620%and 800%higher activity,respectively,than the tac promoter,which has previously been used for gene overexpression in R.sphaeroides.In addition,a Prsp_7571-derived synthetic promoter library with strengths ranging from 54%to 3200%of that of the tac promoter,was created on the basis of visualization of red fluorescent protein(RFP)expression in R.sphaeroides.Finally,as a demonstration,the synthetic pathway of Q10 was modulated by the selected promoter T334*in JDW-710;the Q10 yield in shake-flasks increased 28%and the production reached 226 mg/L.These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R.sphaeroides-derived MCFs.
