Engineering the C4 pathway of Corynebacterium glutamicum for efficient production of 5-aminolevulinic acid / 生物工程学报

5-aminolevulinic acid (5-ALA) plays an important role in the fields of medicine and agriculture. 5-ALA can be produced by engineered Escherichia coli and Corynebacterium glutamicum. We systematically engineered the C4 metabolic pathway of C. glutamicum to further improve its ability to produce 5-ALA. Firstly, the hemA gene encoding 5-ALA synthase (ALAS) from Rhodobacter capsulatus and Rhodopseudomonas palustris were heterologously expressed in C. glutamicum, respectively. The RphemA gene of R. palustris which showed relatively high enzyme activity was selected. Screening of the optimal ribosome binding site sequence RBS5 significantly increased the activity of RphemA. The ALAS activity of the recombinant strain reached (221.87±3.10) U/mg and 5-ALA production increased by 14.3%. Subsequently, knocking out genes encoding α-ketoglutarate dehydrogenase inhibitor protein (odhI) and succinate dehydrogenase (sdhA) increased the flux of succinyl CoA towards the production of 5-ALA. Moreover, inhibiting the expression of hemB by means of sRNA reduced the degradation of 5-ALA, while overexpressing the cysteine/O-acetylserine transporter eamA increased the output efficiency of intracellular 5-ALA. Shake flask fermentation using the engineered strain C. glutamicum 13032/∆odhI/∆sdhA-sRNAhemB- RBS5RphemA-eamA resulted in a yield of 11.90 g/L, which was 57% higher than that of the original strain. Fed-batch fermentation using the engineered strain in a 5 L fermenter produced 25.05 g/L of 5-ALA within 48 h, which is the highest reported-to-date yield of 5-ALA from glucose.

Characterization of the biosorption of fast black azo dye K salt by the bacterium Rhodopseudomonas palustris 51ATA strain

BACKGROUND: Removal of dyes from wastewater by microorganisms through adsorption, degradation, or accumulation has been investigated. Biological methods used for dye treatment are generally always effective and environmentally friendly. In this study, biosorption of the Fast Black K salt azo dye by the bacterium Rhodopseudomonas palustris 51ATA was studied spectrophotometrically, at various pH (2­10), temperatures (25°C, 35°C, and 45°C) and dye concentrations (25­400 mg L-1). RESULTS: The bacterial strain showed extremely good dye-removing potential at various dye concentrations. IR studies at different temperatures showed that the dye was adsorbed on the bacterial surface at lower temperatures. Characteristics of the adsorption process were investigated by Scatchard analysis at 25°C and 35°C. Scatchard analysis of the equilibrium binding data for the dye on this bacterium gave rise to linear plots, indicating that the Langmuir model could be applied. The regression coefficients obtained for the dye from the Freundlich and Langmuir models were significant and divergence from the Scatchard plot was observed. CONCLUSION: The adsorption behavior of the dye on this bacterium was expressed by the Langmuir, Freundlich, and Temkin isotherms. The adsorption data with respect to various temperatures provided an excellent fit to the Freundlich isotherm. However, when the Langmuir and Temkin isotherm models were applied to these data, a good fit was only obtained for the dye at lower temperatures, thus indicating that the biosorption ability of R. palustris 51ATA is dependent on temperature, pH, and dye concentration.

Effects of 5-aminolevulinic acid (ALA)-containing supernatants from selected Rhodopseudomonas palustris strains on rice growth under NaCl stress, with mediating effects on chlorophyll, photosynthetic electron transport and antioxidative enzymes

Background: Rice is globally one of the most important food crops, and NaCl stress is a key factor reducing rice yield. Amelioration of NaCl stress was assessed by determining the growth of rice seedlings treated with culture supernatants containing 5-aminolevulinic acid (ALA) secreted by strains of Rhodopseudomonas palustris (TN114 and PP803) and compared to the effects of synthetic ALA (positive control) and no ALA content (negative control). Results: The relative root growth of rice seedlings was determined under NaCl stress (50 mM NaCl), after 21 d of pretreatment. Pretreatments with 1 μM commercial ALA and 10X diluted culture supernatant of strain TN114 (2.57 μM ALA) gave significantly better growth than 10X diluted PP803 supernatant (2.11 μM ALA). Rice growth measured by dry weight under NaCl stress ordered the pretreatments as: commercial ALA N TN114 N PP803 N negative control. NaCl stress strongly decreased total chlorophyll of the plants that correlated with non-photochemical quenching of fluorescence (NPQ). The salt stress also strongly increased hydrogen peroxide (H2O2) concentration in NaCl-stressed plants. The pretreatments were ordered by reduction in H2O2 content under NaCl stress as: commercial ALA N TN114 N PP803 N negative control. The ALA pretreatments incurred remarkable increases of total chlorophyll and antioxidative activities of catalase (CAT), ascorbate peroxide (APx), glutathione reductase (GR) and superoxide dismutase (SOD); under NaCl stress commercial ALA and TN114 had generally stronger effects than PP803. Conclusions: The strain TN114 has potential as a plant growth stimulating bacterium that might enhance rice growth in saline paddy fields at a lower cost than commercial ALA.

Efecto de microorganismos con potencial probiótico en la calidad del agua y el crecimiento de camarón Litopenaeus vannamei (Decapoda: Penaeidae) en cultivo intensivo / Microorganisms effect with probiotic potential in water quality and growth of the shrimp Litopenaeus vannamei (Decapoda: Penaeidae) in intensive culture

The use of probiotics has gained acceptance in aquaculture, particularly in maintaining water quality and enhancing growth in organisms. This study analyzed the effect of the commercial (EM TM, Japan) natural product composed by (Rhodopseudomonas palustris, Lactobacillus plantarum, Lactobacillus casei and Saccharomyces cerevisiae) added to the water, in order to determine its effect in water quality, sediment and growth of L. vannamei under intensive culture. The evaluation included three treatments with a weekly addition of EM: i) tanks without probiotics (C), ii) tanks with a dose of 4L/ha (EM1) and iii) tanks with a dose of 10L/ha (EM2). The treatment C was carried out three times, while treatments EM1 and EM2 were carried out four times. A total of 4 350 shrimps were measured for total length and weight, to calculate total and porcentual weight gain, daily weight gain, specific growth rate (TCE), and food conversion factor (FCA); besides, the survival rate was estimated. The use of probiotics allowed a shorter harvest time in treatments EM1 (90d) and EM2 (105d) with relation to the treatment C (120d). Treatments EM1 and EM2 were within the recommended intervals for culture, with respect to treatment C. The use of probiotic bacteria significantly regulated pH (EM1, 8.03±0.33; EM2, 7.77±0.22; C, 9.08±0.35) and reduced nitrate concentration (EM1, 0.64±0.25mg/L; EM2, 0.39±0.26mg/L; C, 0.71mg/L). Water pH mostly explained the variance with respect to the treatments. Treatment EM2 presented the greatest removal of organic matter (1.77±0.45%), whereas the contents of extractable phosphorus increased significantly in treatment EM1 with 21.6±7.99mg/kg and in treatment EM2 with 21.6±8.45mg/kg with control relation (14.3±5.47). The shrimp growth was influenced by dissolved oxygen, salinity and pH in the sediment, establishing that salinity was the most important variable in the weight with a negative association. Treatment EM1 recorded an improved TCE (2.69±0.35%/d) and FCA (1.46±0.20) with relation to the control treatment (TCE, 1.88±0.25%/d; FCA, 2.13±0.48). Survival was significantly greater in treatments containing probiotics with 61±8.76% and 60±10.5% for EM1 and EM2, respectively. This study indicated the positive effect obtained with the use of this commercial probiotic, to improve culture conditions and growth parameters in an intensive culture of L. vannamei.

Los probióticos han ganado aceptación en la acuicultura para mantener la calidad del agua y aumentar el crecimiento de los organismos. En este estudio se analizó el efecto de una mezcla comercial de microorganismos eficientes (EM) (Rhodopseudomonas palustris, Lactobacillus plantarum, Lactobacillus casei y Saccharomyces cerevisiae) sobre la calidad del agua, sedimento y el crecimiento en un cultivo intensivo de camarón L. vannamei. La evaluación consistió en tres tratamientos: i) estanques sin EM (C), ii) estanques con dosis de 4L/ha (EM1) y iii) estanques con dosis de 10L/ha (EM2). Los resultados demostraron menor tiempo de cosecha en los tratamientos EM1 (90d) y EM2 (105d). Los tratamientos EM1 y EM2 mantuvieron significativamente regulados los valores del pH (EM1, 8.03±0.33; EM2, 7.77±0.22) y redujeron las concentraciones de nitrato (EM1, 0.64±0.25mg/L; EM2, 0.39±0.26mg/L). El tratamiento EM2 presentó la mayor remoción de materia orgánica (1.77±0.45%). El tratamiento EM1 mejoró la TCE (2.69±0.35%/d) y FCA (1.46±0.20). Los tratamientos EM1 y EM2 presentaron mayor supervivencia con 61±8.76% y 60±10.5%, respectivamente. Este estudio demostró el efecto benéfico del uso de la mezcla comercial en los parámetros ambientales y de crecimiento en un cultivo intensivo de L. vannamei.

Bacteriocin from Purple Nonsulfur Phototrophic Bacteria, Rhodobacter capsulatus

To find whether productivity of bacteriocin is controlled between different species under unusual cultural conditions, we used Rhodobacter capsulatus ATCC 17016 as a producer and Rhodopseudomonas palustris ATCC 17003 as an indicator. Rhodobacter capsulatus was cultured under aerobic conditions in the dark in Lascelles medium containing 0.3% Triton X-100. As a result, bacteriocin productivity increased enormously. The optimal pH range of bacteriocin production was 6~7.8. Through partial purification of bacteriocin, the molecular weight was roughly estimated at 14 kDa. Plasmid had no influence on bacteriocin production by Rhodobacter capsulatus. Our findings indicate that culture conditions affect bacteriocin productivity between more distantly related species, and bacteriocin of Rhodobacter capsulatus is not encoded by a plasmid.

In vitro assessment of gastrointestinal viability of two photosynthetic bacteria, Rhodopseudomonas palustris and Rhodobacter sphaeroides / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The objectives of this study were to assess the potential of two photosynthetic bacteria (PSB), Rhodopseudomonas palustris HZ0301 and Rhodobacter sphaeroides HZ0302, as probiotics in aquaculture. The viability of HZ0301 and HZ0302 in simulated gastric transit conditions (pH 2.0, pH 3.0 and pH 4.0 gastric juices) and in simulated small intestinal transit conditions (pH 8.0, with or without 0.3% bile salts) was tested. The effects of HZ0301 and HZ0302 on the viability and permeability of intestinal epithelial cell in primary culture of tilapias, Oreochromis nilotica, were also detected. All the treatments were determined with three replicates. The simulated gastric transit tolerance of HZ0301 and HZ0302 strains was pH-dependent and correspondingly showed lower viability at pH 2.0 after 180 min compared with pH 3.0 and pH 4.0. Both HZ0301 and HZ0302 were tolerant to simulated small intestine transit with or without bile salts in our research. Moreover, there was no significant difference (P>0.05) among three treatments including the control and the groups treated with HZ0301 or HZ0302 both in intestinal epithelial cell viability and membrane permeability, showing no cell damage. In summary, this study demonstrated that HZ0301 and HZ0302 had high capacity of upper gastrointestinal transit tolerance and were relatively safe for intestinal epithelial cells of tilapias.

The potential use of anoxygenic phototrophic bacteria for treating latex rubber sheet wastewater

A total of 92 isolates of the purple non sulphur photosynthetic bacteria (PNSB) were isolated from 23 samples of wastewater obtained from rubber sheet manufacturing processes from various places of southern, Thailand. The isolate DK6 had the best potential for use in wastewater treatment as it can out-compete indigenous strains of PNSB when grown with them under conditions of microaerobic-light conditions. The isolate DK6 was identified as being most closely allied to Rhodopseudomonas blastica. The optimal pH and temperature for cell growth were between 6.5-7.5 and 30ºC, respectively. Optimum growth of DK6 was obtained after supplementing the wastewater from a latex rubber sheet processing plant with 0.50 percent (NH4)2SO4 and 1 mg/L nicotinic acid under conditions of microaerobic-light (3000 lux). Using these optimum conditions for growth, indigenous microorganisms reduced the initial chemical oxygen demand (COD) of the wastewater from 7,328 to 3371 mg/L a reduction of 54 percent and the biochemical oxygen demand (BOD) (initial BOD 4967 mg/L) by 70 percent. Using the same conditions and either a pure culture of DK6 or a mixed culture (DK6 plus indigenous microorganisms) a reduction of 90 percent of both COD and BOD was achieved. Chemical analysis of the cultures after treatment of the enriched wastewater shows that the protein content of the pure DK6 was 65.2 percent of the dry weight, and in mixed culture the protein content was 66.7 percent. Hence, single cell protein (SCP) may be a possible bi- product of the treatment process.

Production of lipases by four anoxygenic purple non-sulphur phototrophic bacteria.

Production of lipases by Rhodopseudomonas palustris, Rhodobacter sphaeroides, Rhodocyclus gelatinosus and Rhodocyclus tenuis in different synthetic media was investigated. Rc. gelatinosus followed by Rb. sphaeroides were good producers of lipases, while Rps. palustris and Rc. tenuis were poor in lipase secretion. Lipase secretion by Rc. gelatinosus was adaptive in nature, while other three bacterial behavior was inconsistent. No positive correlation could be observed between growth and lipase production.

Photobiodegradation of pyridine by Rhodopseudomonas palustris JA1.

A purple non-sulfur bacterium isolated from dairy effluent was identified as Rps. palustris JA1. This organism was able to grow on pyridine as sole source of carbon in a light dependent anaerobic process with a doubling time of 30 h. Intermediates of pyridine photobiodegradation were identified as glycine and malonate, produced in stoichiometric molar ratios with simultaneous utilization, yielding biomass.

Hydrogen photoproduction from acetate by Rhodopseudomonas palustris / 生物工程学报

Based on the characteristics of metabolism of photosynthetic bacteria and the major kinds of organic compounds produced in wastewater degradation, eleven kinds of organic compounds were chosen for hydrogen photoproduction using Rhodopseudomonas palustris Z strain. The maximal volumetric H2 productivity was obtained using acetate as the sole carbon source and electron donor. The kinetics of cell growth and H2 liberation, and the influences of several major limiting factors on photoevolution of H2 were examined using acetate as carbon source. It was shown that hydrogen production was partially correlated with cell growth. The medium composition of the preculture, the preculture time, and inoculation volume were confirmed to have big effects on hydrogen photoevolution. The time delay of H2 production was evidently shortened using the inoculum of late exponential growth phase or stationary phase using ammonium sulfate as nitrogen source or with the inoculum of middle exponential growth phase using glutamate as the nitrogen source. The identity of temperature and light intensity for H2 evolution and cell growth has significant potential application in the technology of splitting organic acid into H2 by photosynthetic bacteria. The concentrations of acetate and glutamate in the medium affected hydrogen photoevolution and cell growth significantly. The productivity of H2 increased with substrate concentrations when substrate concentrations of sodium acetate and sodium glutamate were lower than 70 mmol/L and 15 mmol/L, respectively. Hydrogen production was inhibited but the cell growth was faster when the concentration of sodium glutamate over 15 mmol/L due to forming free NH4+. The highest rate of hydrogen production was 19.4 mL.L-1.h-1 using 30 mmol/L of sodium acetate as hydrogen donor under the standard conditions, respectively. The optimal conditions for hydrogen production were 35-37 degrees C, 6000-8000 lx and pH 7.3-8.3. The effects of oxygen and inoculation volume on photoproduction of hydrogen were also discussed.

Adaptación de Rhodopseudomonas palustris a cambios de condiciones atmosféricas / Adaptation of Rhodopseudomonas palustris to changes in atmospheric conditions

El burbujeo de O2 95%-N2 5% a un cultivo de Rhodopseudomonas palustris crecido en condiciones fotosintéticas provoca un cese inmediato de la síntesis de bacterioclorofila y una disminución de la actividad de la enzima delta-aminolevulinato sintetasa (ALA-S), sin alterar el crecimiento bacteriano. Al cesar el gaseado el nivel enzimático se recupera rápidamente pero el de bacterioclorofila lo hace más lentamente. El agregado de cloramfenicol al cesar el gaseado, no afecta la recuperación de la actividad enzimática. En cambio, si se lo agrega desde el inicio del gaseado, la actividad igual decae pero su recuperación es menor que en ausencia del antibiótico. La enzima de extractos de células gaseadas muestra activación espontánea a 40-C Se postula el efecto de distintos activadores sobre diversas formas de la enzima ALA-S para explicar estos hechos