Enhanced Biological Hydrogen Production from Escherichia coli with Surface Precipitated Cadmium Sulfide Nanoparticles

The precipitation of cadmium sulfide nanoparticles is induced on the surface of Escherichia coli , and the biological hydrogen production efficiency under visible light (VL) irradiation is investigated. When endogenous [Ni–Fe]‐hydrogenase is anaerobically induced, an additional 400 µmol of hydrogen gas is generated within 3 h from the hybrid system suspension (50 mL) under VL irradiation (2000 W m^?2), corresponding to an increase in hydrogen production of ≈30%. The apparent quantum efficiencies of the hybrid system under 470 and 620 nm VL irradiation are 7.93% and 9.59%, respectively, which are higher than those of many photoheterotrophic bacteria. Furthermore, the mechanism of the enhanced hydrogen evolution is investigated. The interaction between photogenerated electrons and cells of E. coli is confirmed by heat‐treatment, electron‐scavenger, and separation studies. The acceleration of pyruvate generation, inhibition of lactate fermentation, increase of formate concentration, stimulation of hydrogenase activity, and elevation of nicotinamide adenine dinucleotide (NAD)H/NAD ratio in the hybrid system are responsible for the enhanced hydrogen production. A feasibility study is also conducted using wastewater and natural sunlight for the hydrogen production by the hybrid system. An additional 120 µmol of hydrogen is generated from the hybrid system within 3 h under these conditions using natural resources.     

Enhanced Production of Recombinant Proteins in Escherichia coli by Filamentation Suppression

During growth of high-cell-density cultures of Escherichia coli, overproduction of recombinant proteins often results in increased stress response, cell filamentation, and growth cessation. Filamentation of cells consequently lowers final achievable cell concentration and productivity of the target protein. Reported here is a methodology that should prove useful for the enhancement of cell growth and protein productivity by the suppression of cell filamentation. By the coexpression of the E. coli ftsA and ftsZ genes, which encode key proteins in cell division, growth of recombinant strains as well as production of human leptin and human insulin-like growth factor I was improved. Observation of cell morphology revealed that the coexpression of the ftsA and ftsZ genes successfully suppressed filamentation caused by the accumulation of recombinant proteins.     

Extrachromosomal Nature of Hydrogen Sulfide Production in Escherichia coli

Two strains of Escherichia coli which produce hydrogen sulfide appear to have acquired this ability via transfer of genetic material from another genus.     

自生条件下根瘤菌的氢酶表达与固氮的联系

在自生条件下,研究了根瘤菌的氢酶与固氮酶的共轭表达(coexpression)。氢酶表达受碳源限制和氧耗速率的调节,并为固氮酶表达条件促进,在固氮酶放氢的诱导下而与固氮酶共轭表达。此外,观察到外源氢支持根瘤菌自生固氮的活性,氢支持效应被葡萄糖酸钠和氧抑制。     

Contribution of Hydrogenase 2 to Stationary Phase H2 Production by Escherichia coli During Fermentation of Glycerol

Escherichia coli has four hydrogenases (Hyd), three genes of which are encoded by the hya, hyb, and hyc operons. The proton-reducing and hydrogen-oxidizing activities of Hyd-2 (hyb) were analyzed in whole cells grown to stationary phase and cell extracts, respectively, during glycerol fermentation using novel double mutants. H₂ production rate at pH 7.5 was decreased by ~3.5- and ~7-fold in hya and hyc (HDK 103) or hyb and hyc (HDK 203) operon double mutants, respectively, compared with the wild type. At pH 6.5, H₂ production decreased by ~2- and ~5-fold in HDK103 and HDK203, respectively, compared with the wild type. At pH 5.5, H₂ production was reduced by ~4.5-fold in the mutants compared with the wild type. The total hydrogen-oxidizing activity was shown to depend on the pH of the growth medium in agreement with previous findings and was significantly reduced in the HDK103 or HDK203 mutants. At pH 7.5, Hyd-2 activity was 0.26 U (mg protein)^?1 and Hyd-1 activity was 0.1 U (mg protein)^?1. As the pH of the growth medium decreased to 6.5, Hyd-2 activity was 0.16 U (mg protein)^?1, and Hyd-1 was absent. Surprisingly, at pH 5.5, there was an increase in Hyd-2 activity (0.33 U mg protein)^?1 but not in that of Hyd-1. These findings show a major contribution of Hyd-2 to H₂ production during glycerol fermentation that resulted from altered metabolism which surprisingly influenced proton reduction.     

Hydrogenase activity and proton-motive force generation by Escherichia coli during glycerol fermentation

Proton motive force (Δp) generation by Escherichia coli wild type cells during glycerol fermentation was first studied. Its two components, electrical—the membrane potential (?φ) and chemical—the pH transmembrane gradient (ΔpH), were established and the effects of external pH (pH_ex) were determined. Intracellular pH was 7.0 and 6.0 and lower than pH_ex at pH 7.5 and 6.5, respectively; and it was higher than pH_ex at pH 5.5. At high pH_ex, the increase of ?φ (?130 mV) was only partially compensated by a reversed ΔpH, resulting in a low Δp. At low pH_ex ?φ and consequently Δp were decreased. The generation of Δp during glycerol fermentation was compared with glucose fermentation, and the difference in Δp might be due to distinguished mechanisms for H⁺ transport through the membrane, especially to hydrogenase (Hyd) enzymes besides the F₀F₁-ATPase. H⁺ efflux was determined to depend on pH_ex; overall and N,N’-dicyclohexylcarbodiimide (DCCD)-inhibitory H⁺ efflux was maximal at pH 6.5. Moreover, ΔpH was changed at pH 6.5 and Δp was different at pH 6.5 and 5.5 with the hypF mutant lacking all Hyd enzymes. DCCD-inhibited ATPase activity of membrane vesicles was maximal at pH 7.5 and decreased with the hypF mutant. Thus, Δp generation by E. coli during glycerol fermentation is different than that during glucose fermentation. Δp is dependent on pH_ex, and a role of Hyd enzymes in its generation is suggested.     

南岭黄檀根瘤固氮酶和吸氢酶活性研究

测定不同环境条件下南岭黄檀根瘤固氮酶和吸氢酶活性。结果表明,南岭黄檀根瘤具有吸氢酶活性,外源Ｈ＿２可提高固氮酶活性（２５％）,表明吸氢酶有助于提高固氮效率。低浓度的硝酸盐（２０ｍｇ。１￣＿（－１）不影响根瘤固氮酶和吸氢酶活性,但浓度达４０ｍｇ·ｌ￣（－１）时有抑制作用,而铵盐在上述浓度下均表现抑制作用。离体根瘤固氮酶和吸氢酶活性表达的最适温度为２５℃,过高或过低均有抑制作用。土壤含水量及光照强度均明显影响其固氮酶和吸氢酶活性。     

Aerobic Hydrogen Production via Nitrogenase in Azotobacter vinelandii CA6

The diazotroph Azotobacter vinelandii possesses three distinct nitrogenase isoenzymes, all of which produce molecular hydrogen as a by-product. In batch cultures, A. vinelandii strain CA6, a mutant of strain CA, displays multiple phenotypes distinct from its parent: tolerance to tungstate, impaired growth and molybdate transport, and increased hydrogen evolution. Determining and comparing the genomic sequences of strains CA and CA6 revealed a large deletion in CA6''s genome, encompassing genes related to molybdate and iron transport and hydrogen reoxidation. A series of iron uptake analyses and chemostat culture experiments confirmed iron transport impairment and showed that the addition of fixed nitrogen (ammonia) resulted in cessation of hydrogen production. Additional chemostat experiments compared the hydrogen-producing parameters of different strains: in iron-sufficient, tungstate-free conditions, strain CA6''s yields were identical to those of a strain lacking only a single hydrogenase gene. However, in the presence of tungstate, CA6 produced several times more hydrogen. A. vinelandii may hold promise for developing a novel strategy for production of hydrogen as an energy compound.     

Extracellular Iron Reduction Is Mediated in Part by Neutral Red and Hydrogenase in Escherichia coli

Both microbial iron reduction and microbial reduction of anodes in fuel cells can occur by way of soluble electron mediators. To test whether neutral red (NR) mediates iron reduction, as it does anode reduction, by Escherichia coli, ferrous iron levels were monitored in anaerobic cultures grown with amorphous iron oxide. Ferrous iron levels were 19.4 times higher in cultures fermenting pyruvate in the presence of NR than in the absence of NR. NR did not stimulate iron reduction in cultures respiring with nitrate. To explore the mechanism of NR-mediated iron reduction, cell extracts of E. coli were used. Cell extract-NADH-NR mixtures had an enzymatic iron reduction rate almost 15-fold higher than the chemical NR-mediated iron reduction rate observed in controls with no cell extract. Hydrogen was consumed during stationary phase (in which iron reduction was detectable) especially in cultures containing both NR and iron oxide. An E. coli hypE mutant, with no hydrogenase activity, was also impaired in NR-mediated iron reduction activity. NR-mediated iron reduction rates by cell extracts were 1.5 to 2 times higher with hydrogen or formate as the electron source than with NADH. Our findings suggest that hydrogenase donates electrons to NR for extracellular iron reduction. This process appears to be analogous to those of iron reduction by bacteria that use soluble electron mediators (e.g., humic acids and 2,6-anthraquinone disulfonate) and of anode reduction by bacteria using soluble mediators (e.g., NR and thionin) in microbial fuel cells.     

Enhanced Trehalose Production Improves Growth of Escherichia coli under Osmotic Stress

The biosynthesis of trehalose has been previously shown to serve as an important osmoprotectant and stress protectant in Escherichia coli. Our results indicate that overproduction of trehalose (integrated lacI-P_tac-otsBA) above the level produced by the native regulatory system can be used to increase the growth of E. coli in M9-2% glucose medium at 37°C to 41°C and to increase growth at 37°C in the presence of a variety of osmotic-stress agents (hexose sugars, inorganic salts, and pyruvate). Smaller improvements were noted with xylose and some fermentation products (ethanol and pyruvate). Based on these results, overproduction of trehalose may be a useful trait to include in biocatalysts engineered for commodity chemicals.     

深红红螺菌吸氢酶缺失突变株在管式光合反应器中的产氢

本研究设计了一种2 L分体式管式光合反应器, 并研究了深红红螺菌(Rhodospirillum rubrum)吸氢酶缺失突变株在该反应器中分别利用人工光源(持续光照与光暗交替)和自然光的产氢规律。结果表明在人工光照条件下R. rubrum的产氢可维持5 d, 持续光照和光暗交替条件下(12 h: 12 h)的氢产量可分别达到5752 mL/PBR ± 158 mL/PBR和5012 mL/PBR ± 202 mL/PBR; 自然光条件下, 最适产氢光照强度为30000 Lux~40000 Lux; 在此光照条件下, R. rubrum产氢可维持6 d~ 10 d, 最高氢产量可达到2800 mL/PBR。尽管利用自然光的氢产量比利用人工光源氢产量低, 但是利用自然光的产氢比较经济, 并且该光合产氢系统操作简单, 该工艺有望开发为低成本的光合细菌产氢技术。     

Production L-tryptophan by Escherichia coli cells

Whole cells of Escherichia coli B 10 having high tryptophan synthetase activity were used directly as an enzyme source to produce L-tryptophan from indole and L- or D,L-serine. This strain is tryptophan auxotrophic, which is tryptophanase negative and, in addition, L- and D-serine deaminase negative under production conditions. To avoid inhibition of tryptophan synthetase by a high concentration of indole, nonaqueous organic solvents, Amberlite XAD-2 adsorbent, and nonionic detergents were used as reservoirs of indole in the reaction mixture for the production of L-tryptophan. As a result, different effects were observed on the production of L-tryptophan. Particularly, among the nonionic detergents, Triton X-100 was very efficient. Using Triton X-100 for production of L-tryptophan from indole and L- or D,L-serine by whole cells of Escherichia coli B 10, 14.14 g/100 mL and 14.2 g/100 mL of L-tryptophan were produced at 37 degrees C for 60 h.     

Restoration of Escherichia coli hyd B Mutant Hydrogenase by Cultivation in Media Containing Various Metals

Hydrogenase restoration of Escherichia coli hydrogenase deficient mutant HK7, which carries a mutation at hyd B locus, was studied. Anaerobic growth of HK7 in the presence of iron chloride or vanadium chloride resulted in the restoration of hydrogen uptake activity of hydrogenase, but not hydrogen evolution activity. The growth of HK7 in the presence of nickel chloride restored total hydrogenase activity (hydrogen uptake and evolution) as Waugh and Boxer (1986) reported. Therefore, the leniency of HK7 hyd B product might permit the transportation and incorporation of iron chloride or vanadium chloride in hydrogenase, resulting in the alteration of hydrogenase activity.     

产姜黄素大肠杆菌工程菌的构建

姜黄素是姜科植物的特征性成分,具有重要的药理活性.文中利用姜黄素生物合成关键酶β-酮酰辅酶A合酶(Diketide-CoA synthase,DCS)基因和姜黄素合酶(Curcumin synthase,CURS)基因构建非天然融合基因DCS::CURS,并将其与4-香豆酰辅酶A连接酶(4-coumarate coen...     

Restoration of Escherichia coli hyd B Mutant Hydrogenase by Cultivation in Media Containing Various Metals

Hydrogenase restoration of Escherichia coli hydrogenase deficient mutant HK7, which carries a mutation at hyd B locus, was studied. Anaerobic growth of HK7 in the presence of iron chloride or vanadium chloride resulted in the restoration of hydrogen uptake activity of hydrogenase, but not hydrogen evolution activity. The growth of HK7 in the presence of nickel chloride restored total hydrogenase activity (hydrogen uptake and evolution) as Waugh and Boxer (1986) reported. Therefore, the leniency of HK7 hyd B product might permit the transportation and incorporation of iron chloride or vanadium chloride in hydrogenase, resulting in the alteration of hydrogenase activity.     

Production of lycopene by metabolically-engineered Escherichia coli

Escherichia coli strain CAR001 that produces β-carotene was genetically engineered to produce lycopene by deleting genes encoding zeaxanthin glucosyltransferase (crtX) and lycopene β-cyclase (crtY) from the crtEXYIB operon. The resulting strain, LYC001, produced 10.5 mg lycopene/l (6.5 mg/g dry cell weight, DCW). Modulating expression of genes encoding α-ketoglutarate dehydrogenase, succinate dehydrogenase and transaldolase B within central metabolic modules increased NADPH and ATP supplies, leading to a 76 % increase of lycopene yield. Ribosome binding site libraries were further used to modulate expression of genes encoding 1-deoxy-d-xylulose-5-phosphate synthase (dxs) and isopentenyl diphosphate isomerase (idi) and the crt gene operon, which improved the lycopene yield by 32 %. The optimal strain LYC010 produced 3.52 g lycopene/l (50.6 mg/g DCW) in fed-batch fermentation.     

Urea Production and Putrescine Biosynthesis by Escherichia coli

Cultures of Escherichia coli B and K-12 produce urea during growth in minimal medium. Results of isotopic labeling experiments were consistent with the sole source of urea being from the conversion of arginine to putrescine. Since E. coli itself has no demonstrable urease activity, the rate of urea production is a measure of the flux through the arginine to putrescine pathway.