Quantum Chemical Studies on Proteins in the Reaction Center of Rhodobacter sphaeroides

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.

Isolation,Identification of Bacillus Thuringiensis/Cereus and Its Enhancement on Protein Wastewater Treatment by Rhodobacter Sphaeroides

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.

A synthetic biological secondary metabolite,Lycogen^TM,produced and extracted from Rhodobacter sphaeroides WL-APD911 in an optimizatioal scale-up strategy

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.

Optimization of Coenzyme Q10Production Procedure from Genetic Engineered Rhodobacter sphaeroides Overexpressing UbiG

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.

Treatment of soybean wastewater by a wild strain Rhodobacter sphaeroides and to produce protein under natural conditions

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%.

Screening and engineering of high-activity promoter elements through transcriptomics and red fluorescent protein visualization in Rhodobacter sphaeroides

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.

Impacts of Fe2+on 5-aminolevulinic acid(ALA)biosynthesis of Rhodobacter sphaeroides in wastewater treatment by regulating nif gene expression

This study aimed to increase bacterial growth and 5-aminolevulinic acid（ALA） biosynthesis of Rhodobacter sphaeroides in wastewater treatment through adding ferrous ion（ Fe2＋ ）. Results demonstrated that Fe2＋ effectively enhanced the biomass production and ALA yield of R. sphaeroides. Moreover, the optimal Fe2＋ dosage was found to be 400 μmol/L, which was associated with the highest biomass of 4015.3 mg/L and maximum ALA yield of 15.9 mg/g-dry cell weight（mg/g-DCW）. Mechanism analysis revealed that Fe2＋ vastly improved Adenosine Triphosphate（ATP） production by up-regulating the nif gene expression, and increasing ATP enhanced the biomass and ALA yield by supplying energy for bacterial growth and ALA biosynthesis, respectively. Correlation analysis showed that the ALA and ATP yields had positive relation with nifA and nifU gene expression. In addition, the nifA and nifU gene expression displayed high consistency of co-transcription at the optimal Fe2＋ dosage.

Bioproduction of D-psicose using permeabilized cells of newly isolated Rhodobacter sphaeroides SK011

D-psicose exits in an extremely small amount in nature and is difficult to be chemically synthesized.Only three bacteria have been used in the biotransformation of D-psicose from D-fructose.In this paper,another bacterium which could convert D-fructose to D-psicose was isolated and identified as Rhodobacter sphaeroides.The process parameters of D-psicose production using permeabilized cells of Rhodobacter sphaeroides SK011 were optimized,including the permeabilization procedure:0.1%(w/v)CTAB,10 min,and reaction conditions:cell concentration,30 g dry cell wt/L;concentration of substrate,50 g/L;40℃,pH 9.0;reaction time,8 h.Under the optimized conditions,the permeabilized cells produced approximately 6.5 g/L D-psicose with a Dpsicose productivity of 0.82 g·L^(-1)·h^(-1).This is the first report of bioproduction of D-psicose using permeabilized cells of Rhodobacter sphaeroides.

Characteristics for the hybrid cell Foaz obtained from the protoplast fusion between yeast and photosynthetic bacteria in soybean processing wastewater

The optimal pH and temperature, maximum specific degradation rate, half rate constant and flocculation rate for the hybrid cell Foaz were measured in the reaction for the degradation of soybean processing wastewater(SPW) in this study. The optimal pH and temperature for Foaz in SPW were the same as those of its parental strains Saccharomyces cerevisiae Y9407 and Rhodobacter sphaeroides P9479, but the flocculation rate, the maximum specific degradation rate and the half rate constant for Foaz were higher than those of its parental strains. The results suggest that the characteristics of the inter kingdom fusant Foaz constructed from the protoplast fusion between the eukaryote cell yeast S. cerevisiae and the prokaryote cell photosynthetic bacteria R. sphaeroides may favor degrading organic pollutant in SPW and removing biomass from the effluent.

Microglia activation mediated by toll-like receptor-4 impairs brain white matter tracts in rats

Microglia activation and white matter injury coexist after repeated episodes of mild brain trauma and ischemic stroke. Axon degeneration and demyelination can activate microglia; however, it is unclear whether early microglia activation can impair the function of white matter tracts and lead to injury. Rat corpus callosum（CC） slices were treated with lipopolysaccharide（LPS） or LPS ＋ Rhodobacter sphaeroides（RS）-LPS that is a toll-like receptor 4（TLR-4） antagonist. Functional changes reflected by the change of axon compound action potentials（CAPs） and the accumulation of β-amyloid precursor protein（β-APP） in CC nerve fibers. Microglia activation was monitored by ionized calcium binding adaptor-1 immunofluorescent stain, based on well-established morphological criteria and paralleled proportional area measurement. Input-output（I/O） curves of CAPs in response to increased stimuli were significantly downshifted in a dose-dependent manner in LPS（0.2, 0.5 and 1.0μg/mL）-treated slices, implying that axons neurophysiological function was undermined. LPS caused significant β-APP accumulation in CC tissues,reflecting the deterioration of fast axon transport. LPS-induced I/O curve downshift and P-APP accumulation were significantly reversed by the pre-treatment or co-incubation with RS-LPS. RS-LPS alone did not change the I/O curve.The degree of malfunction was correlated with microglia activation, as was shown by the measurements of proportional areas. Function of CC nerve fibers was evidently impaired by microglia activation and reversed by a TLP-4 antagonist, suggesting that the TLP-4 pathway lead to microglia activation.