乙酰丙酸和前体物对Rhodobacter sphaeroides 5-氨基乙酰丙酸合成的影响

球形红细菌Rhodobacter sphaeroides光合成培养过程中(接种量2g/L),分别以不同方式加入不同浓度的乙酰丙酸(LA)、前体物(甘氨酸、琥珀酸),发现LA(30mmol/L)分3次加入,甘氨酸(60mmol/L)分2次加入,可使5-氨基乙酰丙酸合成酶(ALAS)的活性(149.1U/mg)提高,而其脱水酶(ALAD)活性(143.4U/mg)降低,使ALA产量提高到33.8mmol/L.

Rhodobacter sphaeroides和Rhodovulum sulidophilum LHII的α和β亚基的交叉作用关系

类球红细菌(Rhodobacter sphaeroides)和嗜硫小红卵菌(Rhodovulum sulidophilum)为不同属的两种光合细菌,前者的捕光系统Ⅱ由pucB、pucA基因编码产生的β亚基和α亚基组装形成,后者的捕光系统Ⅱ由pucsB、pucsA基因编码产生的β亚基和α亚基组装形成.将这两组基因交叉组合,克隆到包含puc启动子的表达载体中,得到两个表达质粒即pRKpucsBpucA和pRKpucBpucsA,然后通过接合转移方法分别转入LHI、LHII和RC缺陷型菌株DD13中,两种接合转移菌株都可以形成捕光系统Ⅱ并进入光合细菌膜系统.

光合细菌Rhodobacter sphaeroides新型表达系统研究进展

利用当前商业化的表达系统生产重组蛋白时,在寄主培养过程中都不能快速实时地检测重组蛋白的表达水平。光合细菌Rb.sphaeroides是研究细菌光合作用和膜蛋白形成的重要模式生物,具有开发成为一种新型表达系统的潜能。介绍了光合细菌Rb.sphaeroides新型表达系统的优势和开发情况。

浑球红细菌Rhodobacter sphaeroides LHS-305腈水解酶基因的克隆及表达

以筛选的产腈水解酶浑球红细菌Rhodobacter sphaeroides LHS-305为出发菌株,根据已知腈水解酶基因保守区域设计简并引物,从总DNA中成功扩增得到腈水解酶基因的部分片段(402 bp)。通过染色体步移扩增片段的上下游序列,经拼接,得到的腈水解酶基因全长为969 bp(在Genebank数据库中的登录号为JN635494)。该基因与已知腈水解酶序列的最高相似性为82%。构建pET28a-nit表达载体,转化大肠杆菌Rosetta(DE3),得到重组菌。SDS-PAGE电泳结果表明该基因在大肠杆菌中成功表达。重组酶对底物3-氰基吡啶催化结果显示了较高的酶活力,具有跟原始菌相同的特性。

光合细菌Rhodobacter Sphaeroides 2.4.1培养条件的优化

运用单因子和响应面方法,以类胡萝卜素为考察对象对Rhodobacter Sphaeroides 2.4.1的培养条件光照强度、温度、接种量进行优化。结果显示:运用响应面方法优化Rhodobacter Sphaeroides 2.4.1的最适条件为光照强度2 495.85lx、培养温度32℃、接种量10.9%。验证实验结果显示响应面方法优化后的类胡萝卜素含量是17.79mg/L,与理论值17.91mg/L相差0.67%,该数值没有超出误差范围,说明运用响应面方法优化Rhodobacter Sphaeroides2.4.1培养条件有效可行。

Advances in the Coenzyme Q10 Biosynthesis Pathway in Rhodobacter sphaeroides and the Enhancement of Coenzyme Q10 Production Based on Metabolic Engineering

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.

Optimization of Coenzyme Q10Production Procedure from Rhodobacter sphaeroides Overexpressing UbiE

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.

The Effects of Different Carbon Sources on the Growth of <i>Rhodobacter sphaeroides</i>

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.

Comparison of the Photosynthetic Characteristics of Two Developmental Stages in Nostoc sphaeroides Kützing (Cyanophyta)

The photosynthetic activities between two main developmental stages, colony and hormogonium, of the edible cyanobacterium Nostoc sphaeroides Kutzing, were compared. Hormogonia have a higher content of chlorophyll than that of colonies. It showed that the ratios of phycocyain （PC）, allophycocyain （APC） and phycoerythrocyanin （PEC） in hormogonia and colonies were different. The room temperature chlorophyll fluorescence, 77 K chlorophyll fluorescence, measurements of PSⅠand PS Ⅱ activities all showed that colony has higher photosynthetic competence than hormogonia. Hormogonia had a higher respiration rate than colony, while their maximum photosynthetic oxygen evolution rates were very close. The responses of hormogonia and colonies to high light illuminations also were different. Both of their oxygen evolution rates decreased quickly with the prolonged high light illumination, but hormogonia can keep relatively higher PSⅡ activity （Fv/Fm） than that of colonies. The results suggested that colony was photosynthetically more competent than hormogonia, while the ability of hormogonia to tolerate high light illumination was higher than that of colony.

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.

Expression of Acetaldehyde Dehydrogenase Gene Increases Hydrogen Production and Acetate Consumption by <i>Rhodobacter sphaeroides</i>

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.

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.

CtrA Is Nonessential for Cell Cycle Regulation in <i>Rhodobacter sphaeroides</i>

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.

Cellular Localization of Gold and Mechanisms of Gold Resistance in <i>Rhodobacter sphaeroides</i>

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.

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.

Preparation and Performance Test of Gexianmi (Nostoc sphaeroides kützing) Moisturizing Lotion

Gexianmi moisturizing lotion was prepared from Gexianmi extract, carbomer, EDTA-Na2, citric acid, glycerin, butanediol, ceramide, cetearyl alcohol, hydrogenated polydecene, shea butter, etc., and skin moisture contents of different treatments were measured. The data were analyzed by variance analysis and multiple comparisons. The results showed that: Gexianmi moisturizing lotion had a better moisturizing effect, which was better than that of anthocyanin moisturizing lotion. Therefore, Gexianmi moisturizing lotion can be used to effectively maintain the moisture of the human skin stratum corneum.

Enhanced Production of Natural Carotenoids from Genetically Engineered Rhodobacter sphaeroides Overexpressing CrtA

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.

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%.

Enhancement of Rhodobacter sphaeroides growth and carotenoid production through biostimulation

Bacillus thuringiensis/cereus L2 was added as a biostimulant to enhance the biomass accumulation and carotenoid yield of Rhodobacter sphaeroides using wastewater as the culturing medium. Results showed that biostimulation could significantly enhance the R. sphaeroides biomass production and carotenoid yield. The optimal biostimulant proportion was 40 μL（about 6.4 × 10^5CFU）. Through the use of biostimulation, chemical oxygen demand removal, R. sphaeroides biomass production, carotenoid concentration, and carotenoid yield were improved by 178%, 67%, 214%, and 70%, respectively. Theoretical analysis revealed that there were two possible reasons for such increases. One was that biostimulation enhanced the R. sphaeroides wastewater treatment efficiency. The other was that biostimulation significantly decreased the peroxidase activity in R. sphaeroides. The results showed that the highest peroxidase activity dropped by 87% and the induction ratio of the RSP_3419 gene was 3.1 with the addition of biostimulant. The enhanced carotenoid yield in R. sphaeroides could thus be explained by a decrease in peroxidase activity.

Photoregulated or Energy Dependent Process of Hormogonia Differentiation in Nostoc sphaeroides Kutzing （Cyanobacterium）

Hormogonium, which was thought to play an important role in the dispersal and survival of these microorganisms in their natural habitats, is a distinguishable developmental stage of heterocystous cyanobacteria. The present study examined the effects of different light conditions and sugars on the differentiation ofNostoc sphaeroides Kiitzing to the hormogonia stage. Results showed that differentiation of hormogonia was light dependent in the absence of sugar, but that close to 100% of cyanobacteria differentiated to hormogonia in the presence of glucose or sucrose, irrespective of the light conditions. This differentiation was inhibited, even in the presence of sugars, upon application of an inhibitor of respiration. Following the testing of different sugars, the effects of different lights were examined. It was found that 5-10μmol·m^-2·s^-1 photon flux density was optimal for hormogonia differentiation. One hundred percent differentiation was obtained with white light irradiation, in contrast with irradiation with green light (80% differentiation) and red light (0-10% differentiation). Although they showed different efficiencies in inducing hormogonia differentiation in N. sphaeroides, the green and red radiation did not display antagonistic effects. When the additional aspect of time dependence was investigated through the application of different fight radiations and an inhibitor of protein synthesis, it was found that the initial 6 h of the differentiation process was crucial for hormogonia differentiation. Taken together, these results show that hormogonia differentiation in N. sphaeroides is either a photoregulated or an energy deoendent orocess.